Reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium 1,2-bis(pentyloxycarbonyl)ethanesulphonate

Endpoint summary

Description of key information

Subacute toxicity was tested with the registered substance in Wistar rats by oral gavage at 0 (vehicle), 100, 300 and 1000 mg/kg bw/day in a supporting 14-day dose range finding and in a key combined repeated dose/reproductive toxicity study (OECD No. 422). The vehicle was distilled water in the 14-day dose range finding and propylene glycol in the OECD 422 study. The NOAEL for local toxicity of the parental generation was 300 mg/kg bw/day (based on local gastric findings). The NOAEL for systemic toxicity of the parental generation was 1000 mg/kg bw/day.

Subacute oral toxicity was tested similar 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 about 260,  510 and 1000 mg/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 on average basis.  These studies did not reveal toxicity, therefore 1% in the diet, corresponding with >= 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:

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.

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 strain 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: 101 g (female rats), 122 g (male rats)
- 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

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 AY: 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: 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 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
- 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: 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
HISTOPATHOLOGY: Yes
ORGAN WEIGHTS: Yes

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:

1 other: % in the diet

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No toxicological findings

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no toxicological findings

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 ofthe Sulfosuccinates Diester Group, including Butanedioic acid, sulfo-, 1,4 -diamyl 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. 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 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 to be ca.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:

short-term repeated dose toxicity: oral

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 September 2020 to 21 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:

2008

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI (Wistar) rats

Details on species / strain selection:

The test system and the number of animals used in the study were in compliance with the relevant OECD No. 422 guideline. The guideline is designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing. Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.
The minimum number of animals was used, corresponding to the regulatory guidelines being followed, but taking into consideration the scientific reliability of the collected information.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, D-97633 Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 10/11 weeks old (females/males) at start of the treatment and 12/13 weeks old (females/males) at mating.
- Weight at study initiation: Males: 382-462 g, females: 232-276 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Fasting period before study: Not specified
- 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. Rodents were housed 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, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch number: A123) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 560 65984 / 713 70882, Expiry date: 30 November 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): Animals received tap water from municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: 8 days.

DETAILS OF FOOD AND WATER QUALITY: 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 are included in the raw data and are archived at the Test Facility.
The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8-25.0℃ (target range: 19-25°C)
- Humidity (%): 28-66% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12/12 (from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES:
From: Start of in life phase: 25 September 2020 (first vaginal smear sampling)
To: End of in life phase: 02 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:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
As agreed with the Sponsor, no correction for purity of the test item was applied during formulation.
The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with manual shaking and a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared fresh every day prior to administration to animals according to stability assessment results of the analytical method validation study (Study code: 20/031-316AN).
Formulations were prepared in clean glass containers. The appropriate amount test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation, approximately 1-hour ultrasound sonication was applied for proper homogenisation. Formulations were stored surrounded with warming-blocks in a closed container with the intention of keeping formulations above ~25°C until use.

VEHICLE: propylene glycol
- Justification for use and choice of vehicle (if other than water): Based on the available information provided by the Sponsor as well as results of a trial formulation performed at the Test Facility, distilled water was selected as vehicle for the Dose Range Finding (DRF) study (Study code: 20/031-220PE). It was noted during the DRF study that the surfactant type test substance (foamy aqueous formulations) caused local respiratory irritation (by reflux or test item present around the upper respiratory tract) which was considered as a cause of the deaths. These clinical findings were considered not to be related to systemic toxicity, rather the characteristic of the formulations with local effects. Based on these data, as well as results of an additional formulation trial with different vehicles, propylene glycol (abbreviated as PG) was selected as vehicle for this study in agreement with the Sponsor, to reduce the foaming of the formulations and irritation effects. PG is considered as being an acceptable vehicle based on the scientific literature and practice of the Test Facility.
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL (dose formulation concentration)
- Amount of vehicle (if gavage): 5 mL/kg bw (dose formulation volume)
- Lot/batch no. (if required): 1920944
- Purity: 99.99%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed at a total of three occasions (during the first and last weeks and midway during the treatment period). Samples were collected immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Analytical Department.
On each sampling occasion, top, middle and bottom duplicate samples were taken from the dedicated test item formulation(s) for concentration and homogeneity measurement, one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken on three occasions from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.
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 three times during the study (during the first and last weeks and approximately midway during the treatment period).
Any samples not required for analysis were 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: 20/031-316AN). 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) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 98% and 102% 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.
Overall, the formulations were considered adequate for the study.

Duration of treatment / exposure:

Dosing of both sexes began after the acclimatisation (8 days) and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
The first day of dosing of each animal was regarded as Day 0.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females not delivering were sacrificed as practical (on the first day of dam’s necropsy).

Frequency of treatment:

daily on a 7 days/week basis, in each morning

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

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. Based on those results, 1000 mg/kg bw/day was selected as the High dose for this study
- Rationale for animal assignment (if not random):
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
Any unused, spare animals were moved back to the stock colony after they were not needed for the study (after the experiment was ended).
Fifty-five male and 65 female Wistar rats were used in the pre-exposure period. At the end of the pre-exposure period, only 48 females showing regular oestrus cycles and 48 males were allocated to treatment groups (12 animals/sex/groups, 4 groups). Animals were assigned to groups before the start of the treatment
- Fasting period before blood sampling for clinical biochemistry: yes, overnight period of food deprivation, in case of females this happened after the litter has been culled.
- Section schedule rationale (if not random): 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.
At the time of termination, body weight and weight of the following organs of all surviving adult animals was determined:
•With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
•With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and organs were retained from all surviving animals:
Gross findings
Adrenal gland
Animal identification (Fixation and preservation only)
Aorta (Aorta thoracic and abdominal)
Brain (7 section according to the NTP recommendations)
Epididymis
Eye with the optic nerve (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Oesophagus
Femur with marrow
Heart (Section including both ventricles and atria, septum with papillary muscle)
Kidney
Large intestine (Caecum, colon and rectum)
Extraorbital lachrymal gland
Harderian gland
Liver (Liver, 3 lobes, left lateral, right medial, caudate)
Lungs with bronchi (Lungs of euthanized animals was infused with formalin; 3 lobes, left, right cranial, right caudal)
Lymph node ( Mandibular and mesenteric)
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin, subcutis with mammary gland (inguinal)
Skeletal muscle (quadriceps)
Small intestine (Duodenum, ileum and jejunum with Peyer’s patches)
Spinal cord (Transverse sections, 3 levels: cervical, thoracic and lumbar)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix)
Vagina
Additionally, thyroid glands from one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 μm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as above) in the Control and High dose groups (at least 5 animals/sex/group),
• any animals found dead or euthanized pre-terminally during the study in all groups,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina
for females) of all animals of the Control and High dose groups, and all females that failed to deliver healthy pups (documented by a memo in the raw data),
• on the spleen in all Control and High Dose animals, as a special request of study pathologist (described in MEMO),
• the additional histology on the non-glandular region of the stomach of the Control, Low and Mid dose animals to determine the appropriate dose level of the NOAEL according to the Amendment 2 to the Study Plan.
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).
- Dose range finding studies: See endpoint record DRF study 20/031-220PE

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General (routine) clinical observations were made once a day*, during the pre-treatment and treatment period in the afternoon (pm).
*Note: No general clinical observations were made on the day of necropsy.
Animals were inspected for signs of morbidity and mortality once per day in the pre-treatment period and twice daily in the treatment period (at the beginning and end of each working day). Any animal (including also all premature decedents) which showed clinical signs considered severe was sacrificed to prevent suffering, cannibalism and/or autolysis, and was processed in the same way as the animals subjected to terminal necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am), before treatment) and on the day of necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Pertinent behavioural changes and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable.
On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition. The delivery time was recorded as when it was considered that all pups had been born; checking of cages was made during each day up until 16:00.

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

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

FOOD 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

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

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy (on Day 28 in males, on PPD 14 in females).
- Anaesthetic used for blood collection: Yes, pentobarbital
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter has been culled).
- How many animals: randomly selected animals (5 males and 6 females/group). Blood smears were prepared for all selected animals but not examined.
- Parameters examined:
RBC: Red Blood Cell (erythrocyte) count
WBC: White Blood Cell (leukocyte) count
Hgb: Haemoglobin concentration, (g/dL)
Hct: Haematocrit (relative volume of erythrocytes)
MCV: Mean Corpuscular (erythrocyte) Volume
MCH: Mean Corpuscular (erythrocyte) Haemoglobin
MCHC: Mean Corpuscular (erythrocyte) Haemoglobin Concentration
RDW: Red Cell (erythrocyte) volume
Plt: Platelet (thrombocyte) count
MPV: Mean Platelet Thrombocyte volume
RETIC %: Reticulocyte count
NE %: Neutrophil
LY %: Lymphocyte
MO %: Monocyte
BA %: Basophil
EO %: Eosinophil
LUC %: Large Unstained Cells
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 (on Day 28 in males, on PPD 14 in females).
- Animals fasted: : Yes (overnight period of food deprivation, in case of females this happened after the litter has been culled).
- How many animals: randomly selected animals (5 males and 6 females/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: Blood Sampling for Thyroid Hormone Analysis
- Time of blood sample collection: For thyroid hormone analysis, blood samples were taken by venepuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
•from up to two pups per litter on PND4,
•from all dams (but preterminal) and at least two pups per litter on PPD 14 (females) / PND13 (pups),
•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.
- Animals fasted: Not specified
- How many animals:
•from up to two pups per litter on PND4,
•from all dams (but preterminal) and at least two pups per litter on PPD 14 (females) / PND13 (pups),
•from all adult males at termination.

URINALYSIS : Yes
- Time schedule for collection of urine: Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours. The evaluation of the urine samples was performed by using of Medi-Test URYXXON® Stick 10 Urinalysis-strips.
- Metabolism cages used for collection of urine: Yes, for approximately 16 hours
- Animals fasted: (overnight period of food deprivation, in case of females this happened after the litter has 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
Functional Observational Battery and locomotor activity measurement was performed in the study.
- Time schedule for examinations: Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 26 am, females on PPD13 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART).
- Dose groups that were examined: Five males and females/group were randomly selected:
- Battery of functions tested:
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 was monitored by vaginal smears daily during the pre-exposure period before the treatments started. Any females that fail 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. 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 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. Presence of nipples/areolae in the PND13 male pups was also recorded.

HISTOPATHOLOGY: Yes
In case microscopic examination was needed for a tissue or organ, the retained tissues and organs required for histopathology (below) was 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 (at least 5 animals/sex/group),
• any animals found dead or euthanized pre-terminally during the study in all groups,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and all females that failed to deliver healthy pups (documented by a memo in the raw data),
• on the spleen in all Control and High Dose animals, as a special request of study pathologist (described in MEMO),
• the additional histology on the non-glandular region of the stomach of the Control, Low and Mid dose animals to determine the appropriate dose level of the NOAEL according to the Amendment 2 to the Study Plan.
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 other information on material and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related noisy respiration was observed sporadic cases in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortality was observed in the study.
One Mid dose female (#3501) was preterminally euthanised on Day 39 (on the day of delivery).
Moderately decreased activity, piloerection, paleness both eyes and both pinna and red liquid was observed on the day of death. The symptoms were not related to the test item administration.
One High dose female (#4502) was found dead on Day 10. Hunched back, piloerection and noisy respiration were recorded before death, gavage error was the cause of death for this animal.

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.
A sporadic, statistically significantly increased body weight gain (p<0.05) was observed in the Low dose female group in PPD 0-4 period and in the Mid dose male group between Day 0 and Day 27. These were considered incidental, not related to the test item administration

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.
In case of Mid dose males, statistically significantly increased food consumption was observed between Day 14-21 and between Day 14-27, but the data were within the historical control range, hence these findings are not considered to be test item related effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related findings were seen in the clinical pathology parameters.
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.
Statistically significantly decreased haematocrit% value (p<0.01) and statistically significantly increased red cell distribution width% (p<0.01), platelet count (p<0.05) and reticulocyte relative% (p<0.05) was detected in the High dose male group. Statistically significantly decreased MCHC (p<0.05), mean platelet volume (p<0.01) was detected in the High dose female group.
Statistically significantly increased basophils relative% (p<0.01) was observed in the Mid dose male group.
Statistically significantly decreased mean platelet volume (p<0.05), basophils relative% (p<0.05) and statistically significantly increased lymphocytes relative% (p<0.05) was detected in the Low dose female group.
All the values were within the historical control range but red cell distribution width in the High dose male group. There is no dose dependency therefore all the findings considered as not test item related adverse effect.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related findings were seen in the clinical pathology parameters.
There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration.
Urea was statistically significantly decreased in the High dose males (p<0.01) and glucose was significantly increased in the Low dose females (p<0.01) when compared to control animals.
The observed values were within the historical control range, therefore the findings are considered as not being test item related adverse effects.

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.
Statistically significantly increased urine volume (p<0.05) and statistically significantly decreased protein (p<0.05) was observed in the High dose male group. Both data were within the historical control range hence those are not considered as test item related effect.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

At the functional observation battery (FOB), locomotor activity measurement, grip strength and foot splay, there were no effects. There were no changes in animal behaviour or general physical condition 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):

No test item-related effects were observed in the organ weights of the test item treated parental animals compared to controls.
There were no statistically significant differences in the terminal body weight of the test item treated males compared to control males. The body-related weight of the adrenals was decreased statistically significantly (by 14.8%) in the Mid dose group compared to Control, but based on the lack of dose response and as there were no similar change in case of the absolute or brain-related weights, this difference was considered as incidental.
Terminal body weights of test item treated females were not significantly different from control females. The absolute weight of the liver was increased statistically significantly (by 13.9% and by 11.2%) in the Mid and High dose group compared to Controls, without histological change.
Furthermore, the absolute and relative to brain weights of thyroid/parathyroid were statistically significantly higher than Control in the Mid dose group by 27.2% (p<0.05) and 29.4% (p<0.05), but the data showed no dose response and were within the historical control range.
The relative to brain weight of ovaries was statistically significantly higher than Control in the Mid dose group by 17.3% (p<0.05) but the data were within the historical control range.
There were no other statistically significant or biologically relevant differences among groups in the weights of organs measured when compared to Controls in females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer, diffuse minimal to mild infiltrates in non-glandular stomach were observed in the High dose males and females. No other adverse test item related systemic macroscopic changes were recorded at necropsy of routine organs/tissues or in any reproductive organs.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer, diffuse minimal to mild infiltrates in non-glandular stomach were observed in the High dose males and females. No other adverse test item related systemic microscopic changes were recorded at histopathology evaluation of routine organs/tissues or in any reproductive organs.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Oestrus cycle evaluation in females:
•Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00 days was observed in the different groups) before starting the treatment period.
•Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.02, 4.00, 4.01 and 4.06 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded once in each group (#1509, #2504, #3501 and #4509), but these were considered as not being a test item related effect.
Prolonged dioestrus was noted for one High dose female (#4502), but this frequency (1/12) was in line with the normal, expected range and did not affect the mating or pregnancy.

Details on results:

-Clinical signs:
No test item systemic toxicity related clinical signs were observed in the study.
Noisy respiration was observed in three Mid dose females (#3502, #3509, #3511) and one High dose male (#4007) on Day 6, on Day 4, from Day 22 to Day 23 and from Day 1 to Day 4, respectively. The same symptom was observed in four High dose females (#4507, #4508, #4509, #4512) from Day 15 to Day 50. It was observed up to 9 consecutive days.
Gasping respiration was observed in one High dose female (#4507) from Day 32 to Day 36.
Piloerection was recorded from Day 32 to Day 36 in one High dose female (#4507).
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.
Red liquid around vulva was observed for one High dose female (#4501) on Day 53. These findings, but red liquid around vulva were considered incidental, related to the test item administration.
-Mortality and morbidity:
No test item related mortality was observed in the study.
One Mid dose female (#3501) was preterminally euthanised on Day 39 (on the day of delivery).
Moderately decreased activity, piloerection, paleness both eyes and both pinna and red liquid was observed on the day of death. The symptoms were not related to the test item administration.
One High dose female (#4502) was found dead on Day 10. Hunched back, piloerection and noisy respiration were recorded before death, gavage error was the cause of death for this animal.
-Body weight and weight changes:
No test item related adverse effect was observed on body weight or body weight gain parameters in any dose groups for either sex.
A sporadic, statistically significantly increased body weight gain (p<0.05) was observed in the Low dose female group in PPD 0-4 period and in the Mid dose male group between Day 0 and Day 27. These were considered incidental, not related to the test item administration.
-Food consumption:
No test item related adverse effect on food consumption was observed in any test item treated groups, either sex.
In case of Mid dose males, statistically significantly increased food consumption was observed between Day 14-21 and between Day 14-27, but the data were within the historical control range, hence these findings are not considered to be test item related effect.
-Haematological findings:
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data.
Statistically significantly decreased haematocrit% value (p<0.01) and statistically significantly increased red cell distribution width% (p<0.01), platelet count (p<0.05) and reticulocyte relative% (p<0.05) was detected in the High dose male group. Statistically significantly decreased MCHC (p<0.05), mean platelet volume (p<0.01) was detected in the High dose female group.
Statistically significantly increased basophils relative% (p<0.01) was observed in the Mid dose male group.
Statistically significantly decreased mean platelet volume (p<0.05), basophils relative% (p<0.05) and statistically significantly increased lymphocytes relative% (p<0.05) was detected in the Low dose female group.
All the values were within the historical control range but red cell distribution width in the High dose male group. There is no dose dependency therefore all the findings considered as not test item related adverse effect.
-Clinical biochemistry findings:
There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration.
Urea was statistically significantly decreased in the High dose males (p<0.01) and glucose was significantly increased in the Low dose females (p<0.01) when compared to control animals.
The observed values were within the historical control range, therefore the findings are considered as not being test item related adverse effects.
-Endocrine findings:
Thyroid Hormone Analysis: Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any of the male adults and of the PND13 pup dose groups.
The thyroid gland weights of the PND13 pups 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 PND13 pups that were ascribed to the test item.
The measurement of the thyroid hormone levels in the PND4 pups and adult females was not performed as it was not deemed necessary by the Study Director.
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:
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 increased urine volume (p<0.05) and statistically significantly decreased protein (p<0.05) was observed in the High dose male group. Both data were within the historical control range hence those are not considered as test item related effect.
-Behaviour (functional findings):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength and landing foot splay.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes). The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Organ weight findings including organ/body weight ratios
• Parental males
No test item-related effects were observed in the organ weights of the test item treated male animals compared to controls.
There were no statistically significant differences in the terminal body weight of the test item treated males compared to control males. The body-related weight of the adrenals was decreased statistically significantly (by 14.8%) in the Mid dose group compared to Control, but based on the lack of dose response and as there were no similar change in case of the absolute or brain-related weights, this difference was considered as incidental.
• 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 significantly different from control females. The absolute weight of the liver was increased statistically significantly (by 13.9% and by 11.2%) in the Mid and High dose group compared to Controls, without histological change.
Furthermore, the absolute and relative to brain weights of thyroid/parathyroid were statistically significantly higher than Control in the Mid dose group by 27.2% (p<0.05) and 29.4% (p<0.05), but the data showed no dose response and were within the historical control range.
The relative to brain weight of ovaries was statistically significantly higher than Control in the Mid dose group by 17.3% (p<0.05) but the data were within the historical control range.
There were no other statistically significant or biologically relevant differences among groups in the weights of organs measured when compared to Controls in females.
-Gross pathological findings:
NON-PREGNANT FEMALES / Parental Generation
One female in the Control group (#1511), three females in the Low dose group (#2507, #2508 and #2509), four females in the Mid dose group (#3505, #3506, #3509 and #3511) and one female in the High dose group (#4507) were non-pregnant in the study.
Necropsy examination did not show any test item related change in the non-pregnant females or their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One High dose female (#4502) was found dead on Day 10 because of gavage error.
No test item-related macroscopic changes were observed at necropsy, but the perforation of thoracic oesophagus and small thymus were major findings.
PRE-TERMINAL EUTHANASIA / Parental Generation
One Mid Dose female (3501) was preterminally euthanized on Day 39, on the day of delivery.
No test item-related changes were observed at necropsy. Necropsy revealed diffuse pale discolouration of the liver, enlarged ovaries and enlargement/red multifocal discoloration of uterus+cervix-horn.
TERMINAL EUTHANASIA / Parental Generation
Test item-related diffuse/focal/multifocal thickness of the non-glandular stomach mucosa were observed at a dose level of 1000 mg/kg bw/day. Affected animals included 12/12 males and 10/10 females from the High Dose groups.
All other changes were incidental or a common background.
-Histopathological findings: non-neoplastic:
NON-PREGNANT FEMALES / Parental Generation
One female in the Control group (#1511), three females in the Low dose group (#2507, #2508 and #2509), four females in the Mid dose group (#3505, #3506, #3509 and #3511) and one female in the High dose group (#4507) were non-pregnant in the study.
No microscopic changes were observed in the sex or accessory organs/tissues of non-pregnant females or their male mating pairs.
FOUND DEAD ANIMAL / Parental Generation
One High dose female (#4502) was found dead on Day 10 because of gavage error.
Histopathology revealed correlates in the oesophagus including minimal focal perforation and degeneration/necrosis of tunica muscularis, and moderate decrease of cortical lymphocytes in thymus. Other noteworthy microscopic changes included mild decrease of white pulp in the spleen and diffuse minimal hypertrophy of zona fasciculata in adrenals. Thymic splenic and adrenal changes were considered as typical responses to stress. Other microscopic changes were incidental or agonal/post mortal.
PRE-TERMINAL EUTHANASIA / Parental Generation
One Mid Dose female (3501) was preterminally euthanized on Day 39, on the day of delivery.
Microscopic examination showed focal minimal erosion/ulcer of the glandular stomach. Since similar test item-related lesions were observed in terminal High Dose animals, this microscopic change was regarded as test item-related effect. Due to minimal focal distribution, this lesion had probably minor attribution to the clinical conditions of this animal. Histopathological correlates for the macroscopic changes were observed in the liver including minimal centrilobular necrosis, minimal bilateral hypertrophy of corpora lutea in ovaries and mild luminal dilatation of the uterus+cervix-horn. In addition, inflammatory infiltrate in the oviduct, mild increased mucification of the vagina, moderate extramedullary haematopoiesis of the spleen and minimal decrease of cortical lymphocyte in thymus, were seen. Minimal liver necrosis could contribute to the decreased activity of this animal.
TERMINAL EUTHANASIA / Parental Generation
Test item-related findings were observed in the non-glandular stomach at a dose level of 1000 mg/kg bw/day. These findings correlated with changes observed at necropsy.
Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer (superficial erosions confined to the epithelial surface mostly seen), diffuse minimal to mild infiltrates (mixed cell/mixed mononuclear/eosinophilic/neutrophilic) in non-glandular stomach were observed. A total incidence of these findings appeared proportionally between terminal High Dose males and females.
Other microscopic changes were considered incidental or a common background.
The spleen was histologically examined in all Control and High Dose animals due to statistical differences in relative reticulocytes (%) for the High Dose males seen in haematology. No histopathological correlates were observed in the extramedullary haematopoiesis (EMH). The severity and incidence of splenic EMH seen in Control and High Dose males and females, did not indicate any effect of administered test item.
-Other: Oestrus cycle evaluation in females:
• Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 4.00 days was observed in the different groups) before starting the treatment period.
• Exposure period (pre-mating and mating periods)
No indication of test item related effect was seen in the oestrus cycle data, collected during the pre-mating and mating periods (mean cycle length was 4.02, 4.00, 4.01 and 4.06 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded once in each group (#1509, #2504, #3501 and #4509), but these were considered as not being a test item related effect.
Prolonged dioestrus was noted for one High dose female (#4502), but this frequency (1/12) was in line with the normal, expected range and did not affect the mating or pregnancy.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects parental generation

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local gastric effects

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no significant findings

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

A GLP Validation of the Analytical Method Study of Reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium-1,2-bis(pentyloxycarbonyl)ethanesulphonate (EC 941-224-7)

Test Facility Study Code: 20/031-316AN

The purpose of this study was to validate a High Performance Liquid Chromatography method with UV detection (HPLC-UV) in order to determine the concentration of AEROSOL® AY-100 SURFACTANT in formulations, primarily to support OECD No. 422 study (Study code: 20/031-220P).

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 (20/031-316AN)

Selectivity	No interfering component was observed with the control matrices
Reinjection repeatability (7 injections)	RSD% ≤ 1.6%
Linear range	10 – 1000 μg/mL
Limit of Quantification of the method (LOQ)	10 μg/mL
Theoretical quantification limit from the vehicle	~20 μg/mL
Recovery of the test item from vehicle (~1 and ~250 mg/mL in ultrapure water)	103%
Recovery of the test item from vehicle (~1 and ~250 mg/mL in 1,2-propanediol)	95 and 97%
Precision of formulations from vehicle (~1 and ~250 mg/mL in ultrapure water)	0.4 and 0.9%
Precision of formulations from vehicle (~1 and ~250 mg/mL in 1,2-propanediol)	1.7 and 0.4%
Stability of the test item in vehicle (~1 and ~250 mg/mL in ultrapure water) for 5 days at RT	99 and 100%
Stability of the test item in vehicle (~1 and ~250 mg/mL in 1,2-propanediol) for 1 days at RT	101%
Stability of the samples in the autosampler	At least 30 hours
Stock solution stability at 5 ± 3°C	At least 5 days

 

Conclusions:

The NOAEL for local toxicity of the parental generation: 300 mg/kg bw/day. (based on local gastric effects).
The NOAEL for systemic toxicity of the parental generation: 1000 mg/kg bw/day. (based on no significant findings).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium-1,2-bis(pentyloxycarbonyl)ethanesulphonate (CAS 922-80-5, EC 941-224-7) test item following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (propylene glycol).

The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such 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/kg bw/day was selected as the High dose for this study.

 

Experimental design:

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

Results

In summary, under the conditions of this study the daily administration of reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium-1,2-bis(pentyloxycarbonyl)ethanesulphonate (CAS 922-80-5, EC 941-224-7) by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality.

Test item related noisy respiration was observed sporadic cases in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract.

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), locomotor activity measurement, grip strength and foot splay, there were no effects. There were no changes in animal behaviour or general physical condition 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 PPD14.

There were no adverse 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.

Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer, diffuse minimal to mild infiltrates in non-glandular stomach were observed in the High dose males and females. No other adverse test item related systemic 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/kg bw/day.
(based on local gastric effects).

The NOAEL for systemic toxicity of the parental generation: 1000 mg/kg bw/day.

(based on no significant findings).

The NOAEL for reproductive effects of the parental generation: 1000 mg/kg bw/day.

(based on no significant findings).

The NOAEL for Pup development and survival: 1000 mg/kg bw/day.

(based on no significant 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:

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 (5/sex/group) by oral gavage at 0 (distilled water), 100, 300 and 1000 mg/kg bw for up to 14 days in order to determine the dose levels for a subsequent OECD No. 422 study (Szalóki, 2021). All dose formulations were homogenous. One male given 300 mg/kg bw/day and one male dosed at 1000 mg/kg bw/day were found dead. The cause of death was unknown. Noisy and/or laboured respiration was observed in the Mid and High dose groups (males>females). Hunched back was observed in the High dose female group. Decreased activity, hunched back/piloerection and liquid faeces were observed in the High dose male group, but were probably secondary to respiratory distress. In the first week lower weight gain was seen in Mid and High dose male animals, but with a good recovery in the second week. Terminal body weights in the High dose male group were significantly lower than controls. There were no clearly adverse findings in haematology, clinical chemistry or in organ weights. Diffuse/multifocal/focal thickness of the non-glandular mucosa was seen in the High dose groups (males/females) and in the Mid dose males during necropsy. This is indicative of local irritation, but the food intake in the second week of the study was normal. In conclusion, based on this 14-day Dose Range Finding (DRF) study where Reaction mass of sodium (methylbutyl and pentyl) sulfonate and sodium-1,2-bis(pentyloxycarbonyl)ethanesulphonate (EC941-224-7) test item was administered by oral gavage to Wistar rats for 14 consecutive days, the 1000 mg/kg bw/day dose level seemed 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 (propylene glycol), 100, 300 and 1000 mg/kg bw/day (Szalóki, 2022). Based on the results of the DRF study, 1000 mg/kg bw/day was selected as the High dose for this study. 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 item did not result in test item related mortality. Test item related noisy respiration was observed sporadic cases in a few animals ascribed to reflux or minor exposure to test item in the region of the upper respiratory tract. 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), locomotor activity measurement, grip strength and foot splay, there were no effects. There were no changes in animal behaviour or general physical condition in the control or test groups. No test item-related findings were seen in the clinical pathology parameters. Minimal to marked multifocal squamous cell hyperplasia and minimal to mild erosion/ulcer, diffuse minimal to mild infiltrates in non-glandular stomach were observed in the High dose males and females. No other adverse test item related systemic 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/kg bw/day (based on local gastric effects). The NOAEL for systemic toxicity of the parental generation was 1000 mg/kg bw/day (based on no significant findings).

Feeding the test item (Shaffer and Golz, 1957) for 32 days at concentrations of 0.25%; 0.50% and 1 % in the diet of young male albino rats resulted in no significant signs of toxicity. These concentrations are equivalent to mean daily dosages of about 0.26, 0.51 and 1 g act. ingr./kg bw, respectively. Appearance and behaviour of the animals were normal, and at sacrifice and autopsy at the conclusion of the period of feeding, there was no gross pathology that could be attributed to ingestion of the product. Although the study was conducted in male rats only, it was considered as a key study for subacute toxicity testing.

Subchronic and chronic toxicity 

In a key subchronic oral repeated dose toxicity study (Plank et al., 1969) 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 the registered test substance. After 84 days hematological values, blood chemical values and 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. No significant differences in clinical blood chemistry studies and absolute organ weights were 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 to be worst case 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.

Repeated dose inhalation or dermal toxicity

In accordance with column 2 of REACH legislation 2006R1907 Annex IX, these studies did not have to be conducted as repeated dose oral toxicity was investigated, which is an appropriate route of administration for systemic exposure.

General assessment and conclusion

- The NOAEL for systemic toxicity of the parental generation was 1000 mg/kg bw/day in a key subacute rat combined repeated dose/reproductive toxicity study (OECD No. 422). The oral NOAEL of 750 mg/kg bw/day was obtained in the key dietary subchronic toxicity study in rats with registered substance (OECD 408).

- Based on the fact that no relevant target organ changes were seen up to the highest tested doses in the subacute studies (up to 1000 mg act.ingr./kg bw) and that no relevant changes were seen at the dose of 750-1000 mg act. ingr./kg bw in the 90-day toxicity studies, it can be concluded 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 Di-ester category, showing that all substances in the group had a NOAEL of 750-1000 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).