Sodium 1,4-dicyclohexyl sulphonatosuccinate

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 at 0 (distilled water), 100, 300 and 1000/600 mg solid/kg bw/day in a key combined repeated dose/reproductive toxicity study (OECD No. 422). On day 28 of the dosing period the high dose was reduced to 600 mg/kg bw/day due to mortalities and increasing incidence and severity of noisy/laboured respiration.  The NOAEL for local toxicity of the parental generation was 300 mg solid/kg bw/day (based on noisy/laboured respiration at 600 mg solid/kg bw/day). The NOAEL for systemic toxicity of the parental generation was >= 600 mg solid/kg bw/day.

Subacute oral toxicity was tested according to OECD 407 method in male rats at 0.25, 0.5 and 1 % in the diet for 32 days, corresponding with average doses of 240, 510 and 960 mg test material/kg bw. Subchronic oral toxicity was further tested equivalent to OECD 408  method in male and female rats at 1% in the diet for 90 days, corresponding with ca. 750 mg act. ingr./kg bw.  These studies did not reveal toxicity, therefore 1% in the diet, corresponding with ca. 750 mg act. ingr./kg bw can be accepted as NOAEL.

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:

guideline study with acceptable restrictions

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:

no

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: 100g (female rats), 121g (male rats) on average
- 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

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
- Storage temperature of food: Not provided

VEHICLE: No Vehicle

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 A-196: 20 male and 20 female at 1.0 % and 20 male and 20 female as control

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily during the investigation

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

FOOD CONSUMPTION : Yes

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 [No.VI 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: No


OTHER: Organ Weight and Organ to Body and Organ to Brain Weight Ratios

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
ORGAN WEIGHTS: Yes

Other examinations:

Organ Weight and Organ to Body and Organ to Brain Weight Ratios (no effects)

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):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

ca. 750 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No effects up to highest concenrtration

Critical effects observed:

not specified

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.
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 1% of various test items mixed into the diet. The various test items were category members of the Sulfosuccinates Diester Group including Butanedioic acid, sulfo-, 1,4-dicyclohexyl ester, sodium salt. After 84 days 5 hematological values, 4 blood chemical values and 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. 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 findings were noted. Category members of the test item at 1% in the diet (10000 ppm equivalent to 750 mg/kg bw/day) 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 (OECD 422)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 September 2020 to 21 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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, 11/12 weeks old (females/males) at start of the experiment and 13/14 weeks old (females/males) at mating.
- Weight at study initiation: Males: 402-481 g, females: 231-291 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 639 / 609 in Type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027200710, Expiry date: 10 July 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200405, Expiry date: 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. Nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, 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 / 713 70882, Expiry date: 31 October 2020 / 30 April 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: Environmental acclimation period for the study was 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 (H-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis were included in the raw data and were archived at the Test Facility.
The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0-23.7℃ (target range: 19-25°C)
- Humidity (%): 24-76% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, light from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
From: Start of in life phase: 15 September 2020 (first vaginal smear sampling)
To: End of in-life phase: 06 December 2020 (last necropsy)

Route of administration:

oral: gavage

Details on route of administration:

The time of the gavage process was prolonged, to be really sure the rat was well relaxed, and the total amount of gavage liquid was administered slowly into the stomach (‘short’ gavage to the lower oesophagus was avoided).

Vehicle:

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 2.87 was used for this purpose). Concentrations and all dose levels in the study 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 solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were ultrasonicated and stirred with manual shaking and/or a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared fresh every day prior to administration to animals to allow their use according to stability assessment results of the analytical method validation study (Study code: 20/032-316AN).
The appropriate amount of the test item was weighed into a clean, calibrated glass container and then mixed properly (with ultrasonication and manual shaking and/or magnetic stirring) with the calculated amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied as necessary to aid dissolution.
After filling the syringe with the calculated amount to be given to each individual rat, the outside of the gavage tube was cleaned with a wetted tissue first (using the vehicle of the study) and then with a dry tissue, to reduce any potential surface contamination to an absolute minimum. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

VEHICLE: distilled water
- Concentration in vehicle: 0, 20, 60 and 200/120 mg solid/mL
- Amount of vehicle (if gavage): Dose formulation volume = 5 mL/ kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed on four occasions (preferably during the first*, second and last weeks 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: The first analytical occasion (07 October 2020) was repeated on 14 October 2020, due to analytical technical error. The measurements could not be evaluated properly, therefore, the results could not be accurately reported. In agreement with the Study Director, the analytical occasion was repeated following week.
On each sampling occasion, top, middle and bottom duplicate samples was taken from test item formulations for concentration and homogeneity measurement, one set to analyse (which can be collected in replicates as practical) and one set as a back-up. 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 four times during the study (during the first, second and last weeks and once approximately midway during the treatment period).
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 (Study code: 20/032-316AN). 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 RSD (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 95% and 106% of the nominal concentrations. 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:

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; reduced to 600 mg/kg bw/day (actual dose received) at 4 weeks due to adverse clinical effects (local irritation) and mortality.

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: 20/032-220PE, [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), noisy respiration, laboured respiration, increased salivation, decreased activity, rooting of bedding, hunched back and piloerection were recorded. No test item related adverse effect was seen on body weight, food consumption, clinical pathology (including haematology and clinical chemistry). There were changes: e.g. gastrointestinal irritation, but this is no sign of systemic toxicity, therefore, the levels were acceptable. It was considered that 1000 mg solid /kg bw/day dose level was 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 were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day before the first treatment (prior to the start of the treatment).
- Fasting period before blood sampling for clinical biochemistry: Yes, overnight period of food deprivation

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). Animals (including also all premature decedents) which showed clinical signs considered severe were sacrificed to prevent suffering, cannibalism and/or autolysis, and were processed in the same way as the animals subjected to terminal necropsy.

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

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, then on Day 0, and afterwards weekly, and at termination.
Parent females were weighed on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0 (i.e. within 24 hours after parturition), 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD3, GD10, GD17 and PPD10 were measurements only as an aid for the calculation of accurate treatment volumes, these data were 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). Food consumption was measured during mating. Food consumption was measured more frequently during the lactation period (on PPD0, 4, 7, 10 and 13).
Mean daily food consumption per animal was calculated for each interval.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes. Blood smears were prepared for all selected animals but not examined.
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes: Euthanimal 40% (400 mg/mL sodium pentobarbital solution) was used by intraperitoneal injection
- Animals fasted: Yes overnight period of food deprivation, in case of females this happened after the litter had been culled
- How many animals: 5 males and 5 females/group randomly selected.
- Parameters examined:
RBC: Red Blood Cell (erythrocyte) count
WBC: White Blood Cell (leukocyte) count
Hgb: Haemoglobin concentration
Hct: Haematocrit (relative volume of erythrocytes)
MCV: Mean Corpuscular (erythrocyte)
MCH: Mean Corpuscular (erythrocyte) Haemoglobin
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW: Red Cell Distribution width
Plt: Platelet (thrombocyte) count
MPV: Mean Platelet Thrombocyte volume
RETIC %: Reticulocyte count
NE %: Neutrophil
LY %: Lymphocyte
MO %: Monocyte
BA %: Basophil
EO %: Eosinophil
LUC %: Large Unstained Cells

Coagulation:
APTT: Activated Partial Thromboplastin Time
PT Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Animals fasted: Yes overnight period of food deprivation, in case of females this happened after the litter had been culled
- How many animals: 5 males and 5 females/group randomly selected
- Parameters examined:
Glucose: Blood sugar concentration
T-BIL: Total Bilirubin concentration
Urea: Urea concentration
Chol.: Cholesterol concentration
Creat.: Creatinine concentration
Phos.: Phosphorus concentration
Na+ : Sodium concentration
K+: Potassium concentration
Ca++: Calcium concentration
Cl-: Chloride concentration
Tot. Prot. : Total Protein concentration
Alb.: Albumin concentration
A/G: Alb/glob ration
AST/GOT: Aspartate Aminotransferase activity
ALT/GPT: Alanine Aminotransferase activity
GGT: Gamma-Glutamyl transferase activity
ALKP: Alkaline Phosphatase activity
BA: Bile acids

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

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes for approximately 16 hours
- Animals fasted: Yes overnight period of food deprivation, in case of females this happened after the litter had been culled
- Parameters examined:
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 26 am, females on PPD9-10 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART).
- Dose groups that were examined: Five males and five females/group were randomly selected
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
A modified Irwin test was performed when sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals was tested. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. This was repeated 3 times for each animal. The distance between the two resulting ink spots of the hind limbs was measured.
Fore/hind grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data.
Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments was presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

IMMUNOLOGY: No

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

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 were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea was recorded in the females as applicable.
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND13 male pups was also recorded.

HISTOPATHOLOGY: Yes
For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs in the Control and High dose groups (selected 5 animals/sex/group– the same animals as used for Clinical Pathology,
• any animals found dead or euthanized pre-terminally during the study in all groups,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups (this was notified by Memo).
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
No histopathological examination was performed on pups (F1 generation).

Statistics:

See under "Any information on materials and methods incl. tables".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The initial High dose level of 1000 mg solid/kg bw/day was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.

Mortality:

mortality observed, treatment-related

Description (incidence):

Test item related mortality and clinical signs at 1000 mg/kg bw/day. The initial High dose level of 1000 mg solid/kg bw/day was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.
No test item related systemic-toxicity mortality was observed in the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

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:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly decreased red blood count (p<0.01), haemoglobin concentration (p<0.01), haematocrit (p<0.01) and increased red cell distribution width (p<0.05) and relative reticulocyte % (p<0.01) were observed in the High dose male group. These were considered to be related to treatment, although females were normal.
Statistically significantly decreased basophils relative % (p<0.05) in the High and Mid dose male groups and increased platelet volumes in Low (p<0.01) and Mid dose (p<0.05) females were in the normal range and were considered to be sporadic differences unrelated to treatment.
Data of red cell distribution width and haemoglobin concentration was out of the historical control range hence these changes are considered as test item related effects. These changes indicate a very slight anaemia, correlating with increased reticulocytes which indicates compensation. The degree of the changes seen is not sufficient to represent a clearly adverse effect.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no biologically relevant effects on the serum chemistry that could be ascribed to as test item related adverse effect.
Statistically significantly increased sodium level (p<0.05) was observed in the Mid dose male group. Statistically significantly decreased calcium level (p<0.05) and bile acid level was observed in the High dose male (p<0.01) and female (p<0.05) group, but the data were within the historical control range.

Endocrine findings:

no effects observed

Description (incidence and severity):

Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult males.
The thyroid gland weights of the males were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the adult males that were ascribed to the test item.
The measurement of the thyroid hormone levels in the adult females were not performed as it was not deemed necessary by the Study Director.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly decreased pH (p<0.05) was measured in the High dose female group but this finding was within the normal range and not considered as test item related adverse effect. The HC range for pH is 6-7, hence the pH of the other group was outside of the HC range but the High dose female group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

At the functional observation battery (FOB) and locomotor activity measurement, there were no test item related 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

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Parental Males
No test item-related effects were observed in the liver weights of the test item treated male animals compared to controls.
There were no statistically significant differences in the terminal body weight of the test item treated males compared to control males. The body-related and brain-related weight of the liver was increased statistically significantly (by 15.0% and by 11.7%, respectively) in the High dose group compared to Control. Absolute liver was increased by 11.8%, compared to Control, however not statistically significant.
Parental Females
No test item-related effects were observed in the organ weights of the test item treated female animals compared to controls.
Terminal body weights of test item treated females were not different from control females. The brain-related weight of kidneys was increased statistically significantly (by 11.2%) in the Mid dose group compared to control, but there was no dose response and data were within the historical control range, hence this is not considered as test item related effect.
There were no other statistically significant and biologically relevant differences among groups in the weights of organs measured when compared to Controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

NON-PREGNANT FEMALES / Parental Generation: There was one non-pregnant female in the study (#2510). Necropsy examination did not show any test item related change in the non-pregnant females and their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation: One High Dose male (#4001) died on Day 18 and one High Dose female (#4511) died on Day 50 of the Study, laboured and noisy respiration were observed right before the death, hence it is considered that a small amount of test item got into the respiratory system. No test item-related macroscopic changes were observed at necropsy, In the male the stomach and the intestines were dilated with gas, the non-glandular mucosa of the stomach showed multifocal thickness and the lungs were dark red and non-collapsed. In the female the lungs were enlarged and dark red at necropsy.
PRE-TERMINAL EUTHANASIA / Parental Generation: One High Dose female (#4503) was pre-terminally euthanised due to missgavage and laboured/noisy respiration on Day 12 of the Study.
No test item-related macroscopic changes were observed. In the lungs focal dark red discoloration was seen.
TERMINAL EUTHANASIA / Parental Generation: No test item-related macroscopic findings were observed up to the dose level of 1000/600 mg solid/kg bw/day.
All observed changes were considered incidental or a common background.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

NON-PREGNANT FEMALES / Parental Generation: There was one non-pregnant female in the study (#2510). No microscopic changes were observed in the non-pregnant females and the male mating pair.
FOUND DEAD ANIMAL / Parental Generation: One High Dose male (#4001) died on Day 18 and one High Dose female (#4511) died on Day 50 of the Study, laboured and noisy respiration were observed right before the death, hence it is considered that a small amount of test item got into the respiratory system. Microscopically agonal congestion/ hemorrhage was seen in the lungs of the male, in the female increased number of atretic follicles was observed and was considered as incidental. The causes of death could not be determined by histopathology.
PRE-TERMINAL EUTHANASIA / Parental Generation: One High Dose female (#4503) was pre-terminally euthanised due to missgavage and laboured/noisy respiration on Day 12 of the Study. Microscopically acute inflammation of the trachea and decreased cellularity in the thymus was observed and was considered as incidental.
TERMINAL EUTHANASIA / Parental Generation: No test item-related findings were seen at a dose level of 1000 / 600 mg/kg bw/day. The liver weight increase (15% relative to body weight) was not found histologically; the increased weight was ascribed to a normal adaptive hypertrophy, less than is visible histologically. In mutual agreement with the pathologist, no further histopathology analysis was needed at the low and mid dose groups.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure-related or a common background.

Histopathological findings: neoplastic:

no effects observed

Details on results:

-Mortality and morbidity:
No test item related systemic-toxicity mortality was observed in the study.
One High dose female (#4511) found dead on Day 50 (short after delivery) and one High dose male (#4001) found dead on Day 18. One High dose female (#4503) was pre-terminally euthanised on Day 13.
- Clinical observation:
No test item related clinical signs were observed in the Low dose groups.
Laboured respiration (Day 12) and noisy respiration (Day 34) were sporadically observed in animal found dead (#4511). Laboured and noisy respiration were recorded prior to death in animal found dead (#4001). Noisy and laboured respiration was observed prior to death in animal preterminal euthanised (#4503).
Noisy respiration was also observed in case of 1/12 Mid dose male on Day 13 and Day 14, another 3/11 High dose males from Day 3 until Day 26. Noisy respiration was observed for 4/12 Mid dose females from Day 2 to necropsy, the longevity of the observation was 21 days and another 4/12 High dose females from Day 1 to necropsy, the symptom was recorded at a maximum of 15 day long.
Laboured respiration was observed for another 1/12 High dose males on Day 2 and 3 and 2/12 High dose females on Day 12 and from Day 17 to Day 19, respectively.
Piloerection was observed for 1/12 High dose female on Day 2 and Day 3.
The above findings were probably related to the very irritant effect of small amounts of test item reaching the upper respiratory tract via reflux or contamination during dosing (as seen during the preliminary study). This is considered as a local effect of the test item.
Tonic convulsion (whole body) was observed for one Low dose female (#2508) on Day 36, Day 40, Day 43 and Day 46. Nodule around the left axillary was observed for one Mid dose female (#3508) from Day 38 till necropsy, the longevity of the observation was 15 days. These findings were considered incidental, not test item related adverse effect.
-Body weight and weight changes:
No test item related adverse effect on body weight or body weight gain was detected in mean group Low, Mid and High dose animals (males or females).
-Food consumption and compound intake (no feeding study):
No adverse effect on food consumption was seen in male and female animals in any test item treated group. Statistically significantly increased (by 7.8%) mean food consumption was observed for the High dose males from Day 21 to Day 27.
-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 test item related effect of treatment noted in the Irwin test or during the landing foot splay. The average grip strength for the forelimb was statistically significantly increased (p<0.05) in the High dose female group, but the data was within the historical control range and did not follow a dose response.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Haematology:
Statistically significantly decreased red blood count (p<0.01), haemoglobin concentration (p<0.01), haematocrit (p<0.01) and increased red cell distribution width (p<0.05) and relative reticulocyte % (p<0.01) were observed in the High dose male group. These were considered to be related to treatment, although females were normal.
Statistically significantly decreased basophils relative % (p<0.05) in the High and Mid dose male groups and increased platelet volumes in Low (p<0.01) and Mid dose (p<0.05) females were in the normal range and were considered to be sporadic differences unrelated to treatment.
Data of red cell distribution width and haemoglobin concentration was out of the historical control range hence these changes are considered as test item related effects. These changes indicate a very slight anaemia, correlating with increased reticulocytes which indicates compensation. The degree of the changes seen is not sufficient to represent a clearly adverse effect.
-Clinical chemistry:
There were no biologically relevant effects on the serum chemistry that could be ascribed to as test item related adverse effect.
Statistically significantly increased sodium level (p<0.05) was observed in the Mid dose male group. Statistically significantly decreased calcium level (p<0.05) and bile acid level was observed in the High dose male (p<0.01) and female (p<0.05) group, but the data were within the historical control range.
-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.
Statistically significantly decreased pH (p<0.05) was measured in the High dose female group but this finding was within the normal range and not considered as test item related adverse effect. The HC range for pH is 6-7, hence the pH of the other group was outside of the HC range but the High dose female group.
-Organ weights:
> Parental Males: No test item-related effects were observed in the liver weights of the test item treated male animals compared to controls.
There were no statistically significant differences in the terminal body weight of the test item treated males compared to control males. The body-related and brain-related weight of the liver was increased statistically significantly (by 15.0% and by 11.7%, respectively) in the High dose group compared to Control. Absolute liver was increased by 11.8%, compared to Control, however not statistically significant.
> Parental Females: No test item-related effects were observed in the organ weights of the test item treated female animals compared to controls.
Terminal body weights of test item treated females were not different from control females. The brain-related weight of kidneys was increased statistically significantly (by 11.2%) in the Mid dose group compared to control, but there was no dose response and data were within the historical control range, hence this is not considered as test item related effect.
There were no other statistically significant and biologically relevant differences among groups in the weights of organs measured when compared to Controls.
-Pathology evaluation:
>NON-PREGNANT FEMALES / Parental Generation: There was one non-pregnant female in the study (#2510).
Macroscopic Findings: Necropsy examination did not show any test item related change in the non-pregnant females and their male mating pairs.
Microscopic Findings: No microscopic changes were observed in the non-pregnant females and the male mating pair.
>FOUND DEAD ANIMAL / Parental Generation: One High Dose male (#4001) died on Day 18 and one High Dose female (#4511) died on Day 50 of the Study, laboured and noisy respiration were observed right before the death, hence it is considered that a small amount of test item got into the respiratory system.
Macroscopic Findings: No test item-related macroscopic changes were observed at necropsy, In the male the stomach and the intestines were dilated with gas, the non-glandular mucosa of the stomach showed multifocal thickness and the lungs were dark red and non-collapsed. In the female the lungs were enlarged and dark red at necropsy.
Microscopic Findings: Microscopically agonal congestion/ hemorrhage was seen in the lungs of the male, in the female increased number of atretic follicles was observed and was considered as incidental. The causes of death could not be determined by histopathology.
>PRE-TERMINAL EUTHANASIA / Parental Generation: One High Dose female (#4503) was pre-terminally euthanised due to missgavage and laboured/noisy respiration on Day 12 of the Study.
Macroscopic Findings: No test item-related macroscopic changes were observed. In the lungs focal dark red discoloration was seen.
Microscopic Findings: Microscopically acute inflammation of the trachea and decreased cellularity in the thymus was observed and was considered as incidental.
TERMINAL EUTHANASIA / Parental Generation:
Macroscopic Findings: No test item-related macroscopic findings were observed up to the dose level of 1000/600 mg solid/kg bw/day. All observed changes were considered incidental or a common background.
Microscopic Findings: No test item-related findings were seen at a dose level of 1000 / 600 mg/kg bw/day. The liver weight increase (15% relative to body weight) was not found histologically; the increased weight was ascribed to a normal adaptive hypertrophy, less than is visible histologically. In mutual agreement with the pathologist, no further histopathology analysis was needed at the low and mid dose groups.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure-related or a common background.
-Thyroid Hormone Analysis:
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the adult males.
The thyroid gland weights of the males were also statistically not different from the Control group. In summary, there were no effects on the thyroid hormone levels or on the thyroid gland weights in the adult males that were ascribed to the test item.
The measurement of the thyroid hormone levels in the adult females were not performed as it was not deemed necessary by the Study Director.

Key result

Dose descriptor:

NOAEL

Remarks:

local toxicity

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

A GLP Validation of the Analytical Method Study of Dicyclohexyl sodium sulfosuccinate (CAS 23386-52-9; EC 245-629-3)

The purpose of this study was to validate a High Performance Liquid Chromatography method with UV detection (HPLC-UV) in order to determine the concentration (of the active ingredient or the total solid content) of Eurowet CH in formulations, primarily to support OECD No. 422 study (Study code: 20/032-220P).

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

Table 1. Results of the Method Validation (20/032-316AN)

Selectivity	No interfering component was observed with the control matrices
Reinjection repeatability (7 injections)	RSD% ≤ 3.2%
Linear range	50 – 1000 µg/mL of the supplied test item (~17.4 – 348 µg/mL of total solid content)
Limit of Quantification of the method (LOQ)	50 µg/mL of the supplied test item (17.4 µg/mL of total solid content)
Theoretical quantification limit from the vehicle	1 mg/mL of the supplied test item (0.35 mg/mL of total solid content)
Recovery of the test item from vehicle (~3.5 and ~210 mg/mL of total solid in distilled water)	100% and 102%
Precision of formulations from vehicle (~3.5 and ~210 mg/mL of total solid in distilled water)	0.3% and 1.9%
Stability of the test item in vehicle (~3.5 and ~210 mg/mL of total solid in distilled water) for 6 days at room temperature	102% and 103%
Stability of the test item in vehicle (~3.5 and ~210 mg/mL of total solid in distilled water) for 6 days at 5 ± 3°C	100% and 101%
Stability of the samples in the autosampler	At least 80 hours
Stock solution stability at 5 ± 3°C	At least 6 days
Formulation stability at room temperature and at 5 ± 3°C	At least 6 days

 

 

 

Conclusions:

The NOAEL for local toxicity of the parental generation was considered to be 300 mg solid/kg bw/day. (based on noisy/laboured respiration at 600 mg solid/kg bw/day).
The NOAEL for systemic toxicity of the parental generation was considered to be >=600 mg solid/kg bw/day.

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of  the registered substance 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).

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.

Parameters measured during the study included signs of and mortality twice daily, daily general and/or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessments, including a functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. 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 post-natal day (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 and/or offspring. The thyroxine (T4) levels in the Day 13 (PPD13) pups and adult males were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on the results of the DRF study, 1000 mg solid/kg bw/day was selected as the High dose for this study but was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.

Experimental design:

Group Number	Group designation	Dose level (mg solid/kg bw/day)	Dose formulation concentration (mg solid/mL)	Dose formulation volume (mL/kg bw)	Animal numbers
					Male	Female
1	Control	0	0	5	1001-1012	1501-1512
2	Low Dose	100	20		2001-2012	2501-2512
3	Mid Dose	300	60		3001-3012	3501-3512
4	High Dose	1000/600	200/120		4001-4012	4501-4512

Note: On day 28 of the dosing period; the High dose was reduced to 600 mg/kg bw/day due to mortalities and increasing incidence and severity of noisy/laboured respiration at the high dose.

Results:

In summary,  the registered substance given by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 / 600 mg solid/kg bw/day (Low, Mid and High dose groups, respectively) did result in test item related mortality and clinical signs at 1000 mg/kg bw/day.

The initial High dose level of 1000 mg solid/kg bw/day was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality.

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 test item related 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 adverse 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 PPD14.

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

No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs.

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.

The NOAEL for local toxicity of the parental generation: 300 mg solid/kg bw/day. (based on noisy/laboured respiration at 600 mg solid/kg bw/day).

The NOAEL for systemic toxicity of the parental generation was considered to be >=600 mg solid/kg bw/day.

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

The NOAEL for pup development and survival was considered to be >=600 mg solid/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

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 (Szalóki, 2020). All dose formulations were homogenous. Two animals were found dead in the High dose male group (4001 and 4004) during the study. The cause of death was considered not to be related to systemic toxicity, most probably local respiratory irritation from reflux or a very small amount of test item, probably caused the deaths (a known issue with strong surfactants). Noisy respiration, laboured respiration, increased salivation, decreased activity, rooting of bedding, hunched back and piloerection were observed mainly in a few High dose females and males. These changes were not ascribed to systemic toxicity, but probably were secondary to respiratory local effects. No statistically significant test item related effect on body weight, body weight gain or food intake was observed, indicating the local effects of the test item had no major consequences on normal growth. Minor statistically significant changes in red blood cell parameters were not considered as adverse. There were no other biologically relevant haematology or clinical chemistry changes in any of the dose groups compared to the control. In High dose males and females, bodyweight-related liver weights were statistically significantly higher than control; in the absence of clinical pathology changes, this effect was most likely adaptive. At necropsy of early deaths, gastrointestinal irritation was observed as probably test item related. At terminal necropsy local gastric effects indicating local irritation were observed, there were no signs of systemic toxicity. In conclusion, based on this 14-day Dose Range Finding (DRF) study, no clear evidence of systemic toxicity was observed. A dose level of 1000 mg/kg bw/day dose level seems to be 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 (Szalóki, 2021). Based on the results of the DRF study, 1000 mg solid/kg bw/day was selected as the High dose for this study but was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. Parameters measured during the study included signs of and mortality twice daily, daily general and/or weekly detailed observation of clinical signs, weekly body weight and food consumption, and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessments, including a functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. Reproductive performance is reported under Section 7.8. 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 and/or offspring. The thyroxine (T4) levels in the Day 13 (PPD13) pups and adult males were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups.

In summary, the test substance did result in test item related mortality and clinical signs at 1000 mg/kg bw/day. The initial High dose level of 1000 mg solid/kg bw/day was reduced to 600 mg solid/kg bw/day at 4 weeks due to adverse clinical effects (local irritation) and mortality. 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 test item related 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 adverse test item-related findings were seen in the clinical pathology parameters. No test item related macroscopic or microscopic changes were recorded at necropsy or at histopathology evaluation of routine organs/tissues or in any reproductive organs. The NOAEL for local toxicity of the parental generation was 300 mg solid/kg bw/day (based on noisy/laboured respiration at 600 mg solid/kg bw/day). The NOAEL for systemic toxicity of the parental generation was considered to be >=600 mg solid/kg bw/day.

 

Feeding the registered substance (80% purity) for 32 days at concentrations of 0.25, 0.5 and 1 % in the diet of young male albino rats resulted in no significant signs of toxicity (American Cyanamid Company, 1969). These concentrations corresponded with mean test item intake of about 240, 470 and 960 mg/kg bw, respectively. Appearance and behaviour of the animals were normal, and at sacrifice and autopsy at the conclusion of the feeding period, there was no gross pathology that could be attributed to ingestion of the test item. Administration of test item up to 1% in the diet for 32 days in rats did not result in any relevant changes in the subacute toxicity study. Taking into account that the test item contained 80% active ingredient, the highest dose level corresponded with a NOAEL of 768 mg act.ingr./kg bw.

 

Subchronic and chronic toxicity

In a key subchronic oral repeated dose toxicity study with the registered substance, six groups of 40 albino rats (20 males, 20 females) plus 1 control group (20 males, 20 females) were fed with 1% of various test items mixed into the diet . The various test items were category members of the Sulfosuccinates Diester Group including Butanedioic acid, sulfo-, 1,4-dicyclohexyl ester, sodium salt (Plank et al., 1969). After 84 days hematological values, blood chemical values, urinalysis values were measured for all animals, and 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. 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 findings were noted. Administration of category members at 1% in the diet (10000 ppm equivalent to ca.750 mg/kg body weight/day) for 90 days in rats did not result in any relevant changes in the subchronic toxicity study. The NOAEL was therefore considered 750 mg act. ingr./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.

 

General assessment and conclusion

- The NOAEL for systemic toxicity of the parental generation was >= 600 mg solid/kg bw/day in a rat key combined repeated dose/reproductive toxicity study (OECD No. 422).

- The fact that no relevant target organ changes were seen up to highest concentration of 1% in diet for both 32 and 90 days, allows to conclude that corresponding intake of 750 mg/kg bw is NOAEL.

- 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 regulation (No. 1272/2008 of 16 December 2008).