Sodium 1,4-bis(1,3-dimethylbutyl) sulphonatosuccinate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2020 – 22 March 2022 (QA audited draft report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 1,4-bis(1,3-dimethylbutyl) sulphonatosuccinate

EC Number:

219-147-9

EC Name:

Sodium 1,4-bis(1,3-dimethylbutyl) sulphonatosuccinate

Cas Number:

2373-38-8

Molecular formula:

C16H30O7S.Na

IUPAC Name:

sodium 1,4-bis[(4-methylpentan-2-yl)oxy]-1,4-dioxobutane-2-sulfonate

Test material form:

liquid: viscous

Details on test material:

Name: AEROSOL® MA-80 E SURFACTANT
Chemical name: Sodium Dihexyl Sulfosuccinate in mixture of ethanol and water
Active ingredient: Sodium Dihexyl Sulfosuccinate
Total solids: Sodium Dihexyl Sulfosuccinate + its impurities
CAS number: 2373-38-8
EC number: 219-147-9Batch/Lot number: KB20B2201
Description: Clear viscous liquid
Total solids content: 79.7% (based on the CoA)
Conversion factor: 1.255 (corrected to 100%)
Expiry date: 16 February 2021

Test animals

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.
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 experiment and 12/13 weeks old (females/males) at mating.
- Weight at study initiation: Males: 391-441 g, females: 234-282 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 508 in type II, III and/or IV polycarbonate cages. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027200428 / 03027200710, Expiry date: 28 April 2023 / 10 July 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072191028 / 05072200405, Expiry date: 28 October 2022 / 05 April 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Maxi Fun Tunnels, Batch number: 123) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch number: 560 65984 / 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 6 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 the local 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 will be 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): 20.1-25.0℃ (target range: 19-25°C)
- Humidity (%): 32-68% (target range: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES:
From: start of in-life phase: 19 August 2020 (first vaginal smear sampling)
To: End of in-life phase: 02 November 2020 (last necropsy)

Administration / exposure

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 1.255 was applied used for this purpose). Concentrations and all dose levels in the study (including raw data and study report) were expressed as solid matter as requested by the Sponsor.
The test item was formulated in the selected vehicle (distilled water), as a visibly stable homogenous solution at the appropriate concentrations according to the dose levels 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 a maximum of 1 or 7 days* prior to administration to animals according to stability assessment results of the analytical method development and method validation studies (Test Facility Study codes: 20/030-316ANE and 20/030-316AN).
*Note: Maximum of 7 days of storage was applied for 20, 60 and 120 mg solid/mL concentration test item formulations, while test item formulation of 200 mg solid/mL concentration was stored for a maximum of 1 day.
Based on those results of the analytical method validation and its extension, the test item formulation in 5-200 mg test item/mL concentration range (equaling 4-159 mg solid/mL range) were stable for at least 7 days when stored at room temperature, while test item formulation at 300 mg test item/mL concentration (equaling 239 mg solid/mL concentration) was proven to be stable for at least 2 days when stored at room temperature.
The appropriate amount test item was weighed into a clean, calibrated glass container and then mixed properly (with manual shaking and/or magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. Formulations were stored in a closed container at room temperature until use.
After filling the syringe with the calculated amount to be given to each individual rat, the outside of the gavage tube was cleaned with a wetted tissue first (using the vehicle of the study) and then with a dry tissue, to reduce any potential surface contamination to an absolute minimum. A constant volume of 5 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

VEHICLE
- Concentration in vehicle: 0, 20, 60, 120 and 200 mg solid/mL
- Amount of vehicle (if gavage): Dose formulation volume = 5 mL/kg bw
- Lot/batch no. (if required): 202003032 / 202007068

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed at a total of four occasions (during the first* and last weeks and once approximately midway during the treatment period). Samples were collected immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Analytical Department.
*Notes: Sampling was made twice on the first week as the High dose was reduced after three days of treatment: As a formulation with different concentration was used for treatment of the High dose animals from Day 4, additional sampling (sampling #2) was made.
On each sampling occasion, top, middle and bottom duplicate samples were taken from the dedicated test item formulations for concentration and homogeneity measurement, one set to analyse (which was collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken 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 (twice during the first week, once midway during the treatment period and once on the last week of treatment).
Any sample not required for analysis was discarded following acceptance of the results of the formulation analysis by the Contributing Scientist #1 (Analyst) and Study Director.
The formulation analysis was conducted within the determined stability period under the control of the responsible Contributing Scientist #1 (Analyst) in compliance with the analytical method validation and the relevant SOPs of the Test Facility.
Analysis of the formulations for concentration and/or homogeneity of test item was performed using a validated analytical HPLC-UV method (High Performance Liquid Chromatography with ultraviolet detection) in the Analytical Department of the Test Facility by using a validated analytical method (Study code: 20/030-316AN). No density measurement was needed for these aqueous formulation as confirmed by the Contributing Scientist #1 (Analyst).
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the CV (coefficient of variation) of replicates (top, middle and bottom of test item formulations) had to be less than 10%.
The measured concentrations of the test item in the different formulations varied between 94% 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 (6 days) and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 14 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing).

Frequency of treatment:

daily on a 7 days/week basis

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

solid

Doses / concentrations 2

Dose / conc.:

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

Remarks:

solid

Doses / concentrations 3

Dose / conc.:

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

Remarks:

solid;
The dose level for High dose group was 1000 mg/kg bw/day (using 200 mg/mL formulation) for the first three days (on Days 0-2), then from Day 3 it was reduced to 600 mg/kg bw/day (using 120 mg/mL formulation).

No. of animals per sex per dose:

12 animals/sex/group + 2 males and 6 females for replacement purpose in the high dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The oral route was selected as it is one of the possible routes of human exposure as requested by ECHA based on the information provided by the Sponsor (Decision number: CCH-D-2114489558-27-01/F; Helsinki, 14 November 2019). The way of test item and vehicle control item administration was in compliance with the relevant OECD No. 422 guideline.
The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study (Study code: 20/030-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study (after a 14-day treatment period), no mortality occurred in the Control, Low and Mid dose groups (100 and 300 mg solid/kg bw/day, respectively). Two out of the four males and one out of the four females in the High dose group (1000 mg solid/kg bw/day) were found dead during the study; however, the cause of death was considered not to be related to systemic toxicity, rather than local respiratory irritation from reflux or a very small amount of test item (a known issue with strong surfactants). Noisy respiration, laboured respiration, gasping respiration, (increased) salivation, decreased activity, soft faeces, liquid faeces, hunched back, red discharge and piloerection were observed mainly in some of the High dose animals and in one Mid dose animal for a short period. These changes were not ascribed to systemic toxicity, but probably were secondary to respiratory local effects. No 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.
Overall, 1000 mg solid/kg bw/day, was considered as acceptable for the High dose level of this study, with the implementation of additional cleaning of gavage tubes and refined gavage procedures, to reduce the risk of respiratory contamination with test item. Lower doses were spaced with a factor of approximately 3.
Based on the early observations of this study, the dose level of the High dose group was reduced to 600 mg solid/kg bw/day from the fourth day of the 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 at start of the treatment (Day 0).
- Fasting period before blood sampling for clinical biochemistry: All animals including the randomly selected animals for blood sampling were fasted (overnight period of food deprivation, in case of females this happened after the litter had been culled).

Examinations

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 (if any) 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). 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.

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.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes: Euthanimal 40% (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 absolute and %: Reticulocyte count
NE absolute and %: Neutrophil
LY absolute and %: Lymphocyte
MO absolute and %: Monocyte
BA absolute and %: Basophil
EO %: Eosinophil
LUC absolute and %: 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
- Time of blood sample collection:
For thyroid hormone analysis, blood samples were taken by venepuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
from up to two pups per litter on PND4,
from all dams and two pups per litter on PPD14 (females) / PND13 (pups),
from all non-pregnant or not delivered adult females at termination (scheduled / unscheduled),
from all adult males at termination (scheduled / unscheduled).
The collected pup blood (plasma) samples were pooled by litter.
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. Timing was documented in the raw data.
Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4°C). The resulting plasma was divided in at least two aliquots (volume target was at least 150 μL for the first aliquot and at least 75 µL for the second aliquot, any remaining sample (if any) was kept as a third aliquot) and stored in an ultra-freezer (-80±10°C) until analysis

URINALYSIS: Yes
- Time schedule for collection of urine: Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes: 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 23 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 five females/group were randomly selected
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
A modified Irwin test was performed [7] 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 at the first instance (evaluation of forelimbs was not needed).
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 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, if possible. Presence of nipples/areolae in the PND13 male pups was also recorded.

HISTOPATHOLOGY: Yes
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group),
• 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,
• stomach samples of 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.
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".

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The daily administration of Sodium Dihexyl Sulfosuccinate by oral gavage to Wistar rats at a dose level of 100 mg solid/kg bw/day (Low dose group) did not result in test item related clinical signs. In the Mid dose group (300 mg solid/kg bw/day), most animals had minor/transient symptoms of respiratory local effects, related to reflux or by small amounts of test item reaching the upper respiratory tract area in animals; but with no indications of systemic effects of the test item. In the High dose group, the initial dose level of 1000 mg solid/kg bw/day was found to be too high despite very careful gavage procedures to ensure minimal local respiratory effects, so the dose was reduced to 600 mg solid/kg bw/day after three days of treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

The daily administration of Sodium Dihexyl Sulfosuccinate by oral gavage to Wistar rats at a dose level of 100 mg solid/kg bw/day (Low dose group) did not result in test item related mortality. One Mid dose animal died, related to local effects of the test item. In the High dose group, the initial dose level of 1000 mg solid/kg bw/day was found to be too high despite very careful gavage procedures to ensure minimal local respiratory effects, so the dose was reduced to 600 mg solid/kg bw/day after three days of treatment. A relatively high rate of deaths or euthanasia occurred at the High dose level due to the local effects of the test item; necropsy showed local gastric irritation and respiratory effects.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effects were observed on body weight parameters in High dose males and females, this was largely transient, in the first week. In High dose females there was a lower weight gain during gestation (~20% less gain than controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effects were observed on food consumption in High dose males and females, this was largely transient, in the first week.

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 adverse effects were seen in the clinical pathology parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related adverse effects were seen in the clinical pathology parameters.

Endocrine findings:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

no effects observed

Description (incidence and severity):

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

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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

effects observed, treatment-related

Description (incidence and severity):

In animals with respiratory distress, secondary stress effects were recorded (such as organ weight and histological changes in thymus and adrenals).
There was an increase in the hepatic weight (12-14%) in both sexes of the High dose group, indicating an adaptive hypertrophy, but below the level that is detectable histologically.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No 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.
Local toxicity was observed as gastric irritation in the High and Mid dose groups (with minimal incidence at the Low dose).
Gastric reflux resulted in irritation, sometimes severe, in the upper respiratory tract, which was lethal in approximately 50% of the High dose and in one animal in the Mid dose animals.

Neuropathological findings:

not examined

Description (incidence and severity):

Treatment-related findings were observed at the High dose level (600 mg solid/kg bw/day). Stomach (non-glandular), thymus and adrenal gland were identified as target organs. It is considered that gastric irritation was a main finding in animals; thymus and adrenal effects were considered as secondary, stress related effects.

Histopathological findings: neoplastic:

no effects observed

Details on results:

-Mortality and morbidity:
One Mid dose female (#3501 on Day 4) and fourteen High dose animals (#4005 on Day 3, #4008 on Day 18, #4009 on Day 2, #4501 on Day 1, #4502 on Day 5, #4503 on Day 1, #4506 on Day 2, #4507 on Day 15, #4509 on Day 4, #4510 on Day 6, #4512 on Day 12, #4601 on Day 32, #4606 on Day 8 and #4609 on Day 39) were found dead during the study. Three High dose animals (#4012 on Day 23, #4505 on Day 17 and #4602 on Day 9) were pre-terminally euthanized. Thus, a total of 1 Mid dose animal (one female) and 17 High dose animals (4 males and 13 females) were lost during the treatment period.
The indication of gavage associated injury was observed in 3/14 High dose rats (#4506, #4509 and #4512).
No clinical signs were observed in case of #4005, #4009 and #4506.
Gasping and/or noisy respiration or red liquid was observed for 5 High dose females prior to death (#4501, #4502, #4503, #4509, #4512). Laboured, noisy or gasping respiration, piloerection, hunched back and distended abdomen was observed prior to pre-terminal euthanasia in case of #4505 (respiratory signs on Days 4-14, hunched back on Days 5-12, distended abdomen on Day 16) and #4602 animals (respiratory sign on Days 3-8, distended abdomen on Days 7-8). Hunched back, piloerection and noisy or gasping respiration was observed in case of #4507, #4510 and #4609 High dose females prior to death. Noisy respiration and piloerection were observed prior to death for #4601 female. Laboured and noisy respiration, piloerection, hunched back and increased salivation was observed for #4606 female prior to death. Noisy respiration was recorded in one found dead male (#4008) from Day 1 to death (Day 18). In one High dose male (#4012) decreased activity, hunched back, laboured and noisy respiration, piloerection and liquid faeces were recorded, this animal was pre-terminally euthanized due to ethical reason on Day 23.
For one Mid dose female (#3501) gasping and noisy respiration was observed prior to death.
-Clinical observations:
The observed clinical signs for found dead or moribund animals are given in the section on mortality/morbidity. The information below applies the surviving animals.
No clinical signs were observed in the Control male and female and Low dose male animals. Noisy or laboured respiration was recorded for two Low dose females (#2511 on Days 1-2, and #2503 on Day 7).
Laboured respiration was observed in 1/12 Mid dose female (#3510) on Day 38 and Day 39. Noisy respiration was observed occasionally in 8/12 Mid dose males and 10/11 Mid dose females from Day 2 to Day 39 and Day 1 to Day 12 , respectively; longer duration was observed only for one female animal (#3510) in the period of Days 14-31 and on Day 39.
Noisy respiration was observed in 6/10 High dose males and 4/5 High dose females from Day 1 to Day 24 and from Day 0 to Day 59, respectively, the longevity of the observation was 5 and 8 days, respectively. Gasping respiration was observed for one High dose female (#4504) on Days 1-2. Piloerection was observed for two High dose females and two High dose males (#4105 on Day 7 and #4109 on Day 2). Laboured respiration was observed for 3/5 High dose females and for 3/10 High dose males on Day 5 and from Day 29 to Day 34 and on Day 2 and from Day 23 to Day 24, respectively. The longest period of the observation was 3 and 2 days, respectively. Mucoid faeces were observed for one High dose male (#4004) on Day 18. Slightly decreased activity was observed for two High dose males (#4109 from Day 2 to Day 3 and #4010 on Day 21, respectively). Hunched back was observed for two High dose males (#4105 on Day 7 and #4109 on Days 2-3,respectively) and one High dose female (#4508 on Days 17-19).
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 was considered as a local effect of the test item.
-Body weight and weight changes:
Decreased body weight gain or body weight loss was observed in the High dose groups in the first week of treatment, with females recovering to near control weights by Day 14 and males by Day 28. During gestation High dose females gained about 20% less than controls (p<0.05) but weight gain in lactation was normal in all groups although the total weight gain (Day 0 to termination) remained lower for High dose females (p<0.01).
No test item related adverse effects on body weight or body weight gain were detected in Low or Mid dose (100 and 300 mg solid/kg bw/day, respectively) animals (males or females).
Taking account of the necropsy findings, reduced body weight growth and food intake, particularly in the first week, was considered to be related to gastric irritation and/or reflux with respiratory irritation effects.
-Food consumption and compound intake (no feeding study):
Test item related adverse effects on food consumption were observed in High dose females (600 mg solid/kg bw/day) during the pre-mating and gestation phases, no effect was observed in the High dose males or Mid and Low dose groups (300 and 100 mg solid/kg bw/day, respectively).
In case of High dose females, reduced values compared to control were recorded during the pre-mating period (by 25%) and gestation period (by 13%), statistical significance was reached in both cases (p<0.05). Based on the effect on body weight values, the observed values were considered as a test item related adverse effect.
-Neurological assessment:
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength and landing foot splay.
All dose groups of males and females had a normal locomotor activity. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals (males and females) and the Control when evaluating the overall total travelled distance (0-60 minutes), any statistical significance observed occasionally in any 5-minute segment without dose response was considered as incidental. The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Haematology:
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing haematology parameters to the relevant Control data. Occasional statistically significant changes were within the HC range (and with no dose response in some cases), and were considered as biological variability, not related to the test item treatment.
-Clinical Chemistry:
No test item-related adverse changes were detected in the test item treated animals (males and females) when comparing serum chemistry parameters to the relevant Control data.
The statistically significant changes observed in the High dose group (decreased urea and total bilirubin concentration in High dose males, increased urea and decreased chloride concentration in High dose females), had no similar trend in the other sex and were within the historical control range, so were considered as animal variability and not related to the test item treatment.
-Urinalysis:
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
-Organ weights:
There were no test item treatment-related statistically significant differences among groups of males and females in the weights of organs measured when compared to Controls.
Terminal body weights of test item treated males and females were not significantly different from control animals.
The body-related and brain related weight of the liver was statistically significantly increased (by 13.8% and 14.1%) in the High dose males compared to Control, similarly the body related liver weight was statistically significantly increased in the High dose females (by 12.4%) when compared to Control (Table 13). Based on the males and females both having increased liver weight at the High dose with no histological changes, this fact was considered to be a non-adverse adaptive change.
There were no other statistically significant and biologically relevant differences among groups in the weights of organs measured when compared to Controls.
-Pathology evaluation:
FOUND DEAD ANIMALS / Parental Generation
One Mid dose female (3501) and fourteen High dose animals (4005, 4008, 4009, 4501, 4502, 4503, 4506, 4507, 4509, 4510, 4512, 4601, 4606 and 4609) were found dead.
Macroscopic Findings
….
Microscopic Findings
Test item-related multifocal squamous cell hyperplasia, mixed cell submucosal infiltrate (and single occasion of inflammation) and/or erosion/ulcer of the non-glandular stomach mucosa were seen in 10/14 found dead High dose animals. Erosion/ulcer contributed to death in 5/14 High dose animals. A gavage reflux related to the test item induced findings in stomach appeared as possible contributing factors to death in one Mid dose female and six High dose rats. A gavage reflux-related subacute microscopic changes observed in the lungs included aggregation alveolar macrophages, foreign material (bronchiole), mixed/neutrophilic infiltrates and/or granulomatous inflammation (with foreign material) and mixed/neutrophilic infiltrate in trachea. The indication of gavage associated injury (degeneration/necrosis/inflammation of tunica muscularis in the oesophagus) was observed in 3/14 found dead High dose rats. The stomach lesions in unscheduled deaths were similar in distribution as those seen in animals that survived to the end of the study. At necropsy, these stomach lesions were visualized as discoloration and/or thickness. The severity of erosion/ulcer lesions was higher in found dead animals comparing with terminals.
Decreased cellularity of lymphocytes (cortex) in the thymus and diffuse bilateral cortical hypertrophy of zona fasciculata in adrenals microscopically observed in unscheduled deaths, were considered stress-related findings due to treatment and not a direct toxic effect of the test item. Thymic changes were manifested at necropsy as small thymus.
All other changes were incidental or a common background. The results indicated that gastric and respiratory tract irritation were main factors in the deaths.
…
PRE-TERMINAL EUTHANASIA / Parental Generation
Three High dose animals (#4012, #4505 and #4602) were pre-terminally euthanized.
Macroscopic Findings
Test item-related stomach findings contributing to earlier terminations consisted of squamous cell hyperplasia (and mixed cell submucosal infiltrate) and/or erosion/ulcer of the non-glandular mucosa.
Microscopic Findings
Test item-related stomach findings contributing to earlier terminations consisted of squamous cell hyperplasia (and mixed cell submucosal infiltrate) and/or erosion/ulcer of the non-glandular mucosa. This indicated that gastric irritation was a main factor in the moribund conditions.
…
TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
Test item-related macroscopic findings were observed in the stomach (non-glandular) of Mid and High dose animas (dose levels of 300 and 600 mg solid/kg bw/day, respectively) and in the thymus of the High dose group. These changes correlated with microscopic findings.
Diffuse/multifocal thickness of the non-glandular mucosa was observed in 1/23 terminal Mid dose and 13/15 terminal High dose animals. White multifocal discoloration of the non-glandular mucosa was seen in 1/23 Mid dose animal. Small thymus correlated with histopathology was observed in 1/23 High dose animal.
All other changes were incidental or a common background.
Microscopic Findings
Treatment-related findings were observed at the High dose level (600 mg solid/kg bw/day). Stomach (non-glandular), thymus and adrenal gland were identified as target organs. It is considered that gastric irritation was a main finding in animals; thymus and adrenal effects were considered as secondary, stress related effects.
-Thyroid hormone analysis:
Compared to the control, there were no statistically significant thyroid hormone concentration levels recorded in any dose groups of parental males or PND13 pups.
No relevant changes were noted in the absolute or relative (to body / brain) thyroid weights of the parental male or female animals in any dose groups. No histopathology (microscopic) findings were detected in any High dose males or females.
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 glands in parental males and females and in the PND13 pups that were ascribed to the test item. 

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

gross pathology

2

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

other: solid

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

Target system / organ toxicity

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

Any other information on results incl. tables

A GLP Validation of the Analytical Method of Sodium Dihexyl Sulfosuccinate, CAS 2373-38-8, EC 219-147-9 (20/030-316AN)

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 the active ingredient or the total solid content) of AEROSOL® MA-80 E SURFACTANT in formulation, primarily to support OECD No. 422 study (Study code: 20/030‑220P). A secondary purpose was to determine the stability of the test item in the required formulation under its conditions of use and storage.

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 (20/030-316AN)

Selectivity	No interfering component was observed
Reinjection repeatability (7 injections)	RSD%≤ 1.9%
Linear range	20 – 1000 µg/mL of the test item (~15.9 – 796.8 µg/mL of total solid content)
Limit of Quantification	20 µg/mL of the test item (~15.9 µg/mL of total solid content) [º0.2 mg/mL (test item inultrapure water)]
Recovery of thetest itemfrom ultrapure water (5 and 200 mg/mL)	104 and 99%
Precision in ultrapure water (5 and 200 mg/mL)	1.6 and 2.1%
Stability of thetest itemin ultrapure water at 5 and 200 mg/mL at room temperature (1 day)	97 and 99%
Stability of thetest itemin ultrapure water at 5 and 200 mg/mL at room temperature (4 days)	99 and 105%
Stability of thetest itemin ultrapure water at 5 and 200 mg/mL at room temperature (7 days)	100 and 104%
Stability of thetest itemin ultrapure water at 5 and 200 mg/mL at 2-8 °C (4 days)	100 and 108%
Stability of the calibration samples in the autosampler at 50 and 500 µg/mL	107 and 100%; At least 37 hours in the autosampler
Stock solution stability	At least 4 days at 5±3 °C

 

Applicant's summary and conclusion

Conclusions:

The NOAEL for local toxicity of the parental generation: 100 mg solid/kg bw/day (based on local gastric effects).
The NOAEL for systemic toxicity of the parental generation: 300 mg solid/kg bw/day (Although body weight/food consumed and mortality in the High dose group were ascribed to secondary effects of the test item from the direct local effects; there could theoretically also be a potential systemic effect. Taking a conservative approach 300 mg solid/kg bw/day was applied here).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of Sodium Dihexyl Sulfosuccinate (CAS 2373-38-8, EC 219-147-9) test item following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (distilled water).

The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 13 post-partum.

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on those results, 1000 mg/kg bw/day was selected as the High dose for this study. Based on the results of the first three days of dosing (mortalities in the High dose group), the dose level of the High dose group was reduced to 600 mg/kg bw/day. Concentrations and all dose levels in the study (including raw data and study report) were expressed as solid matter as requested by the Sponsor.

Experimental design:

Group Number	Group designation	Dose level (mg solid/kg bw/day)	Dose formulation concentration (mg solid/mL)	Dose formulation volume (mL/kg bw)	Number of animals
					Male	Female
1	Control	0	0	5	12	12
2	Low dose	100	20		12	12
3	Mid dose	300	60		12	12
4	High dose*	1000 / 600*	200 / 120*		14	18

*Notes: The dose level for High dose group was 1000 mg/kg bw/day (using 200 mg/mL formulation) for the first three days, then it was reduced to 600 mg/kg bw/day (using 120 mg/mL formulation). Replacement animals started on Day 4 or after (#4602, 4609 and 4610) were treated only at 600 mg/kg bw/day. Dose level of the High dose group is shown as 600 mg/kg bw/day in the report text and tables.

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The thyroxine (T4) levels in the PND13 pups and parental males were also determined.

For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, all found dead or
pre-terminally euthanized animals, and stomach samples of all animals.

Dosing formulation were analysed for concentration and/or homogeneity on four occasions during the study. All test item formulations were shown to be homogeneous. The measured concentrations of the test item in the different formulations varied between 94% and 102% of the nominal concentrations. Overall, the formulations were considered adequate for the study.

RESULTS

In summary, under the conditions of this study the daily administration of Sodium Dihexyl Sulfosuccinate by oral gavage to Wistar rats at a dose level of 100 mg solid/kg bw/day (Low dose group) did not result in test item related mortality or clinical signs. In the Mid dose group
(300 mg solid/kg bw/day), most animals had minor/transient symptoms of respiratory local effects, related to reflux or by small amounts of test item reaching the upper respiratory tract area in animals; but with no indications of systemic effects of the test item. One Mid dose animal died, related to local effects of the test item. In the High dose group, the initial dose level of 1000 mg solid/kg bw/day was found to be too high despite very careful gavage procedures to ensure minimal local respiratory effects, so the dose was reduced to 600 mg solid/kg bw/day after three days of treatment. A relatively high rate of deaths or euthanasia occurred at the High dose level due to the local effects of the test item; necropsy showed local gastric irritation and respiratory effects.

Test item related adverse effects were observed on body weight parameters and food consumption in High dose males and females, this was largely transient, in the first week. In High dose females there was a lower weight gain during gestation (~20% less gain than controls).

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 test item-related adverse effects 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 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. Local toxicity was observed as gastric irritation in the High and Mid dose groups (with minimal incidence at the Low dose). Gastric reflux resulted in irritation, sometimes severe, in the upper respiratory tract, which was lethal in approximately 50% of the High dose and in one animal in the Mid dose animals. In animals with respiratory distress, secondary stress effects were recorded (such as organ weight and histological changes in thymus and adrenals).

There was an increase in the hepatic weight (12-14%) in both sexes of the High dose group, indicating an adaptive hypertrophy, but below the level that is detectable histologically.

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

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

The NOAEL for local toxicity of the parental generation: 100 mg solid/kg bw/day (based on local gastric effects).

The NOAEL for systemic toxicity of the parental generation: 300 mg solid/kg bw/day (Although body weight/food consumed and mortality in the High dose group were ascribed to secondary effects of the test item from the direct local effects; there could theoretically also be a potential systemic effect. Taking a conservative approach 300 mg solid/kg bw/day was applied here).

The NOAEL for reproductive effects of the parental generation: 600 mg solid/kg bw/day (based on no significant findings).

The NOAEL for pups (F1 generation) development and survival: 600 mg solid/kg bw/day (based on no significant findings).