Butanedioic acid, sulfo-, 4-C12-14 (even numbered)-alkyl esters, disodium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (distilled water), 60, 150 and 450 mg/kg bw/day combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD No. 422). The NOAEL for reproductive effects of the parental generation was 450 mg/kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 450 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 February 2021 (study plan) – 26 October 2022 (final report)

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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI (Wistar)

Details on species / strain selection:

Details on species/strain selection:
Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 10/11 weeks old (males/females)
- Weight at study initiation: Males: 372-436 g, females: 233-279 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:
- Housing: Rodents were group-housed, up to 2 animals of the same sex and group/cage in type II, III and/or IV polycarbonate cages. During the mating and gestation, delivery, lactation period, they were paired or individually housed (with pups), respectively. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027201024 / 03027201208, Expiry date: 24 October 2023 / 08 December 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200824, Expiry date: 24 August 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): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch number: 71370882 / 18776795, Expiry date: 30 April
2021 / 31 August 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum. The supplier provided an analytical certificate for the batch used.
- 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: 8 days

DETAILS OF FOOD AND WATER QUALITY:
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 National Public Health and Medical Offer Service (H-8200 Veszprém, József Attila u. 36., Hungary). The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 - 24.6°C (target: 22 ± 3°C)
- Humidity (%): 20 - 75% (target: 30 - 70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 12 February 2021 (start of in life phase) To: 01 May 2021 (last necropsy)

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on 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 (distilled water), as a visibly stable homogenous suspension 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 at 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 development and method validation studies (Study code: 20/125-316ANE and 20/125-316AN). Based on those results, the test item formulation in the 5 and 100 mg/mL concentration range were stable for at least 3 days when stored at room temperature.
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 in a closed container at room temperature until use.

VEHICLE: distilled water
- Concentration in vehicle: 0, 12, 30 and 90 mg/mL for the dose levels group of 0, 60, 150 and 450 mg/kg bw/day, respectively.
- Amount of vehicle (if gavage): A constant volume of 5 mL/kg bw was administered to all animals
- Lot/batch no.: 202011110

Details on mating procedure:

- M/F ratio per cage: 1:1 mating
- Length of cohabitation: Females remained with the same male until copulation occurred, for up to 7 days.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy: A vaginal smear was prepared daily during the mating period and stained with 1% (w/v) aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).
- After successful mating each pregnant female was caged (how): Sperm positive females were caged individually.

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 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.
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 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 at three times during the study (during the first and last weeks and once approximately midway during the treatment period).
The formulation analysis was conducted within the determined stability period.
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: 20/125-316AN). 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 criterion of the concentration analysis was 100 ± 15% of the nominal concentration.
Acceptance criterion of the homogeneity was that the RSD% (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 96.1% and 100.2% of the nominal concentrations, all of those values were within the acceptance criterion (100 ± 15% of the nominal concentration).
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:

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.

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals: not applicable: screening study
- Selection of parents from F1 generation: not applicable: screening study
- Age at mating of the mated animals in the study: 12/13 weeks old (males/females) at mating (Parental generation (P))

Dose / conc.:

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

Remarks:

low dose

Dose / conc.:

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

Remarks:

mid dose

Dose / conc.:

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

Remarks:

high dose

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route was requested by ECHA as the most appropriate route of human exposure (ECHA decision number: CCH-D-2114489557-29-01/F, Helsinki, 14 November 2019; information provided by the Sponsor). 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: 20/125-220PE), 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, the test item was formulated in distilled water and administered by oral gavage to Wistar rats for 14 consecutive days at dose levels of 100, 300 or 1000 mg/kg body weight/day using a dose volume of 8 mL/kg body weight or at dose level of 600 mg/kg body weight/day using the same dose volume for 10 consecutive days. The following findings were recorded in the DRF study:
Mortality / morbidity (a total of four males and four females) was observed in the 1000 mg/kg bw/day dose group within the first four days of treatment.
On Day 7, body weight of the animals in the 600 mg/kg bw/day dose group was significantly lower than controls (-23.6% and -3.1% in males/females). Food consumption on Day 7 was also significantly lower than control (-65.4% and -27.7% versus control). Hunched back, noisy respiration and soft faeces were also observed in this dose group for males and females, while decreased activity, piloerection, partially closed eyelids, incontinence, increased salivation and red discharge (nose/snout) were observed for males of this dose group. Statistically significant changes were observed for increased neutrophils, decreased lymphocytes in males dosed at 600 mg/kg bw/day. The dose at 600 mg/kg bw/day was considered to exceed the MTD (Maximum Tolerated Dose).
On Day 14, body weight of the animals in the 100 (0.4% and -2.6% in males/females) and 300 mg/kg bw/day (-3.7% and -1.1% in males/females) dose groups was not significantly lower than controls. Food consumption day 0-13 was not significantly lower in the males dosed at 100 and 300 mg/kg bw (4.2% and -5.4%) and females dosed at 300 mg/kg bw/day (-4.2%) but it was significant in females dosed at 100 mg/kg bw (-7.2% versus controls). Noisy respiration was observed in the 300 mg/kg bw/day male animals (1/4) and in the 100 mg/kg bw/day females (1/4) as well.
Test item related increase in the weights of adrenal and stomach and decrease in the weights of thymus were observed in the male animals of the 600 mg/kg bw/day dose group; macroscopic changes in adrenal, stomach and thymus correlated with the organ weight changes. A statistically significant increased weights of liver and stomach were observed in female animals of the 600 and 300 mg/kg bw/day dose groups, macroscopic changes only in the stomach with correlating organ weight changes.
Since the dose level of 600 mg/kg bw/day exceeded the MTD and the effects at 300 mg/kg bw/day were less than the effects intended for a High dose level, it was considered that selecting a High dose level of above 450 mg/kg/day for the main study would be likely to cause mortality or severe toxic effects. A High dose of 450 mg/kg bw/day was considered to be suitable for the main 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 v9.3, 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 second week of treatment.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight period of food deprivation, in case of females this happened after the litter had been culled

Parental animals: Observations and examinations:

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. There was no such a case in the study.


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 (on PPD0, 4, 7, 10 and 13).
Main daily food consumption was calculated for each interval..

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

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored by vaginal smears daily for 2 weeks 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).
*Note: Due to difficulties of dose selection the pre-exposure period was extended by one week. As oestrus cycle information were already collected no additional monitoring was made in that last week of pre-exposure period for animal welfare (to reduce stress).
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.

Sperm parameters (parental animals):

For the adult animals, detailed histological examination was performed on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males) of all animals of the Control and High dose groups including the mating pair which failed to deliver healthy pups*.
*Note: Only one non-pregnant female (#4508) was observed in the study. As that female belonged to the High dose group, reproductive organs (and also for her mating pair, #4008) were examined at the first instance.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.
On PND 4, litters were culled to yield 5 males and 5 females per litter (or as nearly as possible). Pups to be culled within each litter were selected randomly. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND0), and on PND4, PND7 and PND13, with accuracy of 0.01 g.
All the litters were checked and recorded daily for the number of viable and dead pups; clinical signs and any abnormal behaviour or appearance of the pups (external abnormalities) were also recorded on each day. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.
On PND 4, litters were culled to yield 5 males and 5 females per litter (or as nearly as possible). Pups to be culled within each litter were selected randomly. In litters of insufficient size where the number of males or female pups was less than 5, adjustment of the selection process was made to assure 10 pups were retained. Culling was not performed on litter sizes less (or equal) than 10. All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). The anogenital distance was also normalized to a measure of pup size (the cube root of body weight) for statistical analysis as considered appropriate by the Study Director.
Number of nipples/areolae in male pups were recorded on PND13.
One male and one female pup per litter (if possible) were previously selected for culling for blood sampling on PND4.
All pups were necropsied on PND13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead
All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not assessed
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not assessed

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals one day after the last treatment
- Maternal animals: All surviving animals one day after the last treatment

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened, and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and of corpora lutea was recorded in the females as applicable.

HISTOPATHOLOGY / ORGAN WEIGHTS
At the time of termination, body weight and weight of the following organs of all surviving adult animals were 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
Adrenals
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
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
•on the selected list of retained tissues and organs (as above) in the Control and High dose groups (selected 5 animals/sex/group),
•one Low dose animal found dead during the study,
•all macroscopic findings (abnormalities), except of minor order from all animals,
•stomach and thymus samples of all the remaining Control, Low, Mid and High dose animals (males / females),
•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 including the mating pair which failed to deliver healthy pups*.
*Note: Only one non-pregnant female (#4508) was observed in the study. As that female belonged to the High dose group, reproductive organs (and also for her mating pair, #4008) were examined at the first instance.
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.

Postmortem examinations (offspring):

SACRIFICE
- Litters were culled on PND4. All selected F1 offspring were terminated on Post-natal Day (PND) 13. In order to allow for overnight fasting of dam prior necropsy on PPD14, offspring was euthanized on PPD/PND13, and the dams on PPD/PND14.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: All culled pups were subjected to necropsy with detailed macroscopic external and internal examination for any abnormalities.

GROSS NECROPSY
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 / ORGAN WEIGTHS
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.

No histopathological examination was performed on pups (F1 generation).

Statistics:

see "Any other information on materials and method incl. tables"

Reproductive indices:

-Male Mating Index (Measure of male’s ability to mate): (Number of males with confirmed mating / Total Number of males cohabited) x 100
-Female Mating Index (Measure of female’s ability to mate): (Number of sperm-positive females / Total Number of females cohabited) x 100
-Male Fertility Index (Measure of male’s ability to produce sperm that can fertilise eggs): (Number of males impregnating a female / Total Number of males cohabited) x 100
-Female Fertility Index (Measure of female’s ability to become pregnant): (Number of pregnant females / Number of sperm-positive females) x 100
-Gestation Index (Measure of pregnancy that provides at least one live pup): (Number of females with live born pups / Number of pregnant females) x 100

Offspring viability indices:

-Survival Index %: [Number of live pups (at designated time) / Number of pups born] x 100
Survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born.
-Pre-implantation mortality %: [(Number of Corpora lutea – Number of implantations) / (Number of Corpora lutea)] x 100
-Intrauterine mortality %: [(Number of implantations – Number of liveborns) / Number of implantations] x 100
-Total mortality %: [(Number of implantations-Number of viable pups (PND0/4/13)) /Number of implantations] x 100
-Sex ratio% (females): [Number of female pups (PND0/4/13) / Number of viable pups (PND0/4/13)] x 100

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the study.
Noisy respiration was detected for a High dose male (#4002) on Days 24-25.
A nodule (1-2 cm) was observed for one Control female (#1502) in the abdominal area from Day 38 until termination.
Alopecia was observed on both forelimbs of another Control female (#1510) from Day 38 until termination.
These findings were considered incidental, not related to the test item administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item related mortality was observed in the study. One Low dose male (#2011) was found dead on Day 23, no clinical signs were shown by this animal prior to death. This case was considered incidental, not related to the test item administration as no test item-related macroscopic or microscopic changes were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect was observed on body weight parameters in High dose (450 mg/kg bw/day) males. No test item related adverse effect on body weight or body weight gain was detected in High dose females, or in the Mid or Low dose (150 or 60 mg/kg bw/day, respectively) animals (males or females).
In the first week of treatment, the High dose males had almost no growth, compared to Control males. The difference in body weight compared to control reached statistical significance by the end of the treatment period (body weight lower by 7% at p<0.05 and body weight gain lower by 47% at p<0.01). These results of the High dose males were considered as a test item related adverse effect (and suitable for a High dose in an OECD No. 422 study).
No effect on body weight parameters was seen in Mid and Low dose males.
No clearly adverse effect on body weight parameters was seen in the test item treated females when compared to concurrent controls. The High dose weight gain was lower than the pre-treatment period and lower than typical historical control data for the pre-mating period, although in this study the control weight gain was similar to the High dose. The observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period, gestation or lactation in all dose groups.
No effect on body weight parameters was seen in Mid and Low dose females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect on food consumption was observed in High dose males
(450 mg/kg bw/day) during the treatment period, no effect was observed in the High dose females, or Mid and Low dose groups (150 and 60 mg/kg bw/day, respectively) of both sexes.
In case of High dose males, reduced values compared to control were recorded during the entire treatment period, statistical significance reached (p<0.01). Based on the body weight values, the observed values were considered as a test item related adverse effect (probably secondary to gastric irritation caused by the test item).
No effect on food consumption was considered in Mid and Low dose male animals. Although the mean food consumption calculated for the entire treatment period in the Mid dose males was statistically significantly lower than Control (by 5.6%, p<0.01), but the difference originated from a transient reduced food consumption of the first week (no statistically significant differences were observed on the other three weeks thereafter), this it was considered as a palatability issue, not being a test item related effect.
No statistically significant effect was observed in test item treated female animals. Although slightly lower than control-level food consumption was recorded for High dose females during the pre-mating (11% below control in the first week), gestation (~5% for GD0-GD20) or lactation period (6.2% during the last part of the lactation period i.e. maximal milk production period), although the differences were not great, the trends indicate some effect which may be below the level considered as adverse.

Haematological findings:

no effects observed

Description (incidence and severity):

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. The sporadic statistical differences in the ratio of reticulocytes and eosinophils as well as the prothrombin time in High dose males were not outside the normal control range, based on historic control data.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.
Increased bile acid concentration was observed in High dose males (by >300%, at p<0.01) and High dose females (by 65%, statistically not significant), although a statistically significant increase was also seen in Low dose males (by >100%, at p<0.01) without dose response . The differences between groups are within the ranges seen in similar study control groups, the dose response was not consistent, there were no adverse histological hepatic findings or any other evidence in clinical pathology for hepatic effects of the test item; hence the apparent elevation in some groups is not considered to reflect an adverse effect of treatment.

Endocrine findings:

no effects observed

Description (incidence and severity):

No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis.

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. Occasional differences with statistical significance but without dose response were considered as animal variability.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

-FOUND DEAD ANIMAL / Parental Generation (Low dose male (#2011) was found dead on Day 23): Microscopic Findings: Microscopically, mild congestion correlated with the macroscopic findings in the liver and lungs. Minimal extramedullary haematopoiesis was observed in the spleen. The histopathological factor contributing to the death of this found dead animal was not determined.
- TERMINAL EUTHANASIA / Parental Generation: Microscopic Findings: Test item-related findings were observed in the stomach and thymus in both sexes.
Stomach: Minimal to moderate focal erosion/ulcer of the non-glandular stomach was observed in 5/12 High dose males. Mild to marked multifocal/diffuse squamous cell hyperplasia of the non-glandular stomach was observed in 12/12 High dose males, and at minimal to mild severity in 7/11 High dose females. 5/12 Mid dose males had minimal multifocal squamous hyperplasia of the non-glandular stomach and in 5/12 Mid dose females at minimal or mild severity. The incidence of minimal squamous cell hyperplasia of the non-glandular stomach in 2/12 Low dose females is considered comparable to the incidence of 1/12 in Control females and not related to test item administration. Submucosal focal/multifocal, mixed/neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 6/12 High dose males. Mucosal focal/multifocal neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 3/12 High dose males.
Thymus: Minimal reduced cellularity of the cortex was observed in the thymus in 3/12 High dose males and 4/11 High dose females. In the Mid dose females reduced cellularity of the thymus was observed in 4/12 animals. The incidence of minimal decreased cellularity in 2/12 Low dose females is considered as within the normal background.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure-related or a common background.
- NON-PREGNANT FEMALES / Parental Generation (one high dose female #4508): microscopic findings: Organs from the reproductive system (ovary, oviduct, uterus, cervix and vagina from female; testes, epididymis, prostate, seminal vesicles and coagulating glands from males) were microscopically examined from the non-pregnant High dose female and its mating partner but did not reveal any test item-related changes.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

-Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 3.97-4.07 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 4.00, 4.06, 4.03 and 4.09 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded for one Low dose (#2507) and one High dose (#4511) female, but as it did not affect mating or pregnancy and as the incidence was sporadic, this fact was considered as being an occasional finding, not being a test item related effect.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. Test item administration was considered to have no impact on the duration of the mating period.

-mortality and morbidity:
No test item related mortality was observed in the study.
One Low dose male (#2011) was found dead on Day 23, no clinical signs were shown by this animal prior to death. This case was considered incidental, not related to the test item administration as no test item-related macroscopic or microscopic changes were observed.
-clinical observations:
No test item related clinical signs were observed in the study.
Noisy respiration was detected for a High dose male (#4002) on Days 24-25.
A nodule (1-2 cm) was observed for one Control female (#1502) in the abdominal area from Day 38 until termination.
Alopecia was observed on both forelimbs of another Control female (#1510) from Day 38 until termination.
These findings were considered incidental, not related to the test item administration.
-body weight and body weight gain:
Test item related adverse effect was observed on body weight parameters in High dose (450 mg/kg bw/day) males. No test item related adverse effect on body weight or body weight gain was detected in High dose females, or in the Mid or Low dose (150 or 60 mg/kg bw/day, respectively) animals (males or females).
In the first week of treatment, the High dose males had almost no growth, compared to Control males. The difference in body weight compared to control reached statistical significance by the end of the treatment period (body weight lower by 7% at p<0.05 and body weight gain lower by 47% at p<0.01). These results of the High dose males were considered as a test item related adverse effect (and suitable for a High dose in an OECD No. 422 study).
No effect on body weight parameters was seen in Mid and Low dose males.
No clearly adverse effect on body weight parameters was seen in the test item treated females when compared to concurrent controls (Table 2). The High dose weight gain was lower than the pre-treatment period and lower than typical historical control data for the pre-mating period, although in this study the control weight gain was similar to the High dose. The observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period, gestation or lactation in all dose groups.
No effect on body weight parameters was seen in Mid and Low dose females.
-food consumption:
Test item related adverse effect on food consumption was observed in High dose males
(450 mg/kg bw/day) during the treatment period, no effect was observed in the High dose females, or Mid and Low dose groups (150 and 60 mg/kg bw/day, respectively) of both sexes.
In case of High dose males, reduced values compared to control were recorded during the entire treatment period, statistical significance reached (p<0.01). Based on the body weight values, the observed values were considered as a test item related adverse effect (probably secondary to gastric irritation caused by the test item).
No effect on food consumption was considered in Mid and Low dose male animals. Although the mean food consumption calculated for the entire treatment period in the Mid dose males was statistically significantly lower than Control (by 5.6%, p<0.01), but the difference originated from a transient reduced food consumption of the first week (no statistically significant differences were observed on the other three weeks thereafter), this it was considered as a palatability issue, not being a test item related effect.
No statistically significant effect was observed in test item treated female animals. Although slightly lower than control-level food consumption was recorded for High dose females during the pre-mating (11% below control in the first week), gestation (~5% for GD0-GD20) or lactation period (6.2% during the last part of the lactation period i.e. maximal milk production period), although the differences were not great, the trends indicate some effect which may be below the level considered as adverse.
-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). 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. The sporadic statistical differences in the ratio of reticulocytes and eosinophils as well as the prothrombin time in High dose males were not outside the normal control range, based on historic control data.
-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.
Increased bile acid concentration was observed in High dose males (by >300%, at p<0.01) and High dose females (by 65%, statistically not significant), although a statistically significant increase was also seen in Low dose males (by >100%, at p<0.01) without dose response
The differences between groups are within the ranges seen in similar study control groups, the dose response was not consistent, there were no adverse histological hepatic findings or any other evidence in clinical pathology for hepatic effects of the test item; hence the apparent elevation in some groups is not considered to reflect an adverse effect of 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. Occasional differences with statistical significance but without dose response were considered as animal variability.
-oestrus cycle evaluation in females
Pre-exposure period
Each female selected for the study showed acceptable cycles (mean cycle length of 3.97-4.07 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 4.00, 4.06, 4.03 and 4.09 days in the Control, Low dose, Mid dose and High dose groups, respectively).
Prolonged oestrus was recorded for one Low dose (#2507) and one High dose (#4511) female, but as it did not affect mating or pregnancy and as the incidence was sporadic, this fact was considered as being an occasional finding, not being a test item related effect.
No prolonged dioestrus or no pseudopregnancy was noted for any females.
-reproductive ability assessment and indices
There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of the test item. The mating index was 100% in all groups. The fertility index was 100% in the Control, Low and Mid dose groups and 92% in the High dose group (males and females). As the value of the High dose group was within the historical control range and in the absence of any microscopic findings, no test item effect was concluded. The gestation index was also 100% in all groups.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred mostly within 5 days of pairing (cohabitation). The mean duration of mating was 3.00, 1.92, 2.17 and 3.33 days in the Control, Low, Mid and High dose groups, respectively. Longer than usual mating was noted for three animals (6 days for #1508 and #1511 and 7 days for #4511), but they were considered as incidental findings, no test item effect was noted.
-evaluation of the gestation, parturition and post-partum period:
There was no effect of treatment noted during the gestation period, parturition or post-partum period in any of dose groups.
The mean duration of pregnancy was comparable in the Control and test item treated groups.
As far as it could be observed during the study, the parturition was normal for all animals.
The number of implantation sites was comparable to the control mean in all dose groups, and no statistically significant differences were noted.
There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose groups.
-organ weights:
Parental Males
Test item-related effect was observed in the thymus weight of High dose males compared to control animals.
Terminal body weights of High dose males were statistically significantly lower than Control (by approx. -8%, the difference was statistically significant at p<0.01). Treatment-related decrease was observed in the thymus weights of Mid and High dose males and there was a microscopic correlate for the weight reduction in High dose males. The thymus changes are very common in stressed animals where markedly reduced body weight growth occurs in the first week of treatment (as in this study); since the effect is considered to be related to body weight and not a direct effect on the thymus, it is not considered as an adverse effect of treatment.
There were statistically significant differences among groups in the weights of a few other organs (brain, heart, seminal vesicle and liver), either in the absolute and/or body weight / brain weight related value when compared to Control. However, in the absence of any macroscopic or microscopic correlate, these weight changes were considered as not related to test item administration.
Parental Females
Test item-related effects were observed in the thymus 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.
Weight differences were observed in the thymus and liver weights. There was macroscopic and microscopic correlate for the thymus weight reduction in females, however, there was no correlate for the weight increase in the liver.
In males a reduced thymus weight and cellularity was ascribed to a general stress effect rather than a direct effect of the test item on the thymus; in High dose females, a clear gastric irritation seen at histology and non-statistical trend for lower food intake are considered to be related to a non-specific stress which is a common cause of these thymic changes. Liver hypertrophy is common in animals treated with xenobiotics, but when the effect is below about 15% (of increase liver weight relative to body weight) the change is usually too small to be detectable by histology examination. The liver weight difference in the absence of histology changes is considered to be non-adverse.
There were no other statistically significant or biologically relevant differences among groups in the weights of organs measured when compared to Controls in females.
-pathology evaluation:
FOUND DEAD ANIMAL / Parental Generation
One Low dose male (#2011) was found dead on Day 23 (no clinical signs were shown by this animal prior to death).
Macroscopic Findings
Necropsy revealed diffuse dark red discolouration and enlargement of all lobes of liver and lungs.
Microscopic Findings
Microscopically, mild congestion correlated with the macroscopic findings in the liver and lungs. Minimal extramedullary haematopoiesis was observed in the spleen. The histopathological factor contributing to the death of this found dead animal was not determined.

TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
Treatment related focal/multifocal thickness of the wall of non-glandular stomach was observed in 9/12 High dose males. Small thymus was observed in 2/11 High dose females.
All other observed changes were considered incidental or a common background.
Microscopic Findings
Test item-related findings were observed in the stomach and thymus in both sexes.
Stomach: Minimal to moderate focal erosion/ulcer of the non-glandular stomach was observed in 5/12 High dose males. Mild to marked multifocal/diffuse squamous cell hyperplasia of the non-glandular stomach was observed in 12/12 High dose males, and at minimal to mild severity in 7/11 High dose females. 5/12 Mid dose males had minimal multifocal squamous hyperplasia of the non-glandular stomach and in 5/12 Mid dose females at minimal or mild severity. The incidence of minimal squamous cell hyperplasia of the non-glandular stomach in 2/12 Low dose females is considered comparable to the incidence of 1/12 in Control females and not related to test item administration. Submucosal focal/multifocal, mixed/neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 6/12 High dose males. Mucosal focal/multifocal neutrophilic inflammatory cell infiltrate was observed in the non-glandular stomach of 3/12 High dose males.
Thymus: Minimal reduced cellularity of the cortex was observed in the thymus in 3/12 High dose males and 4/11 High dose females. In the Mid dose females reduced cellularity of the thymus was observed in 4/12 animals. The incidence of minimal decreased cellularity in 2/12 Low dose females is considered as within the normal background.
All other changes are commonly seen in control and/or treated animals, or were without meaningful differences in severity and incidence, therefore were regarded as incidental, procedure-related or a common background.

NON-PREGNANT FEMALES / Parental Generation
One non-pregnant High dose female was identified in the study (#4508).
Macroscopic Findings
Necropsy examination did not show any relevant test item related change in the non-pregnant female and their male mating pair in the High dose group (only multifocal thickness of the non-glandular stomach was observed in both animals).
Microscopic Findings
Organs from the reproductive system (ovary, oviduct, uterus, cervix and vagina from female; testes, epididymis, prostate, seminal vesicles and coagulating glands from males) were microscopically examined from the non-pregnant High dose female and its mating partner but did not reveal any test item-related changes.

-thyroid hormone analysis
No test item effect was noted in any dose groups based on the results the T4 hormone measurement, thyroid gland weights and histopathology evaluation.
No statistically significant or biologically relevant changes in the T4 thyroid hormone concentration was detected in the test item treated parental males when compared to control.
No relevant changes were noted in the absolute thyroid or relative (to body) thyroid weights of male and female animals in any dose groups. Furthermore, no histopathology (microscopic) findings were detected in any test item treated animals (the bilateral enlargement recorded in a Low dose female (#2505) at necropsy was not confirmed by histopathology).

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: No other findings

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No other findings

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw (total dose)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Description (incidence and severity):

Based on the external evaluation, most of the pups were normal, clinical signs or abnormalities were recorded for a Control litter (#1510), where fur thin was recorded for several pups on PND12 and PND13. These events were considered as minor, incidental findings, not related to the test item treatment.

Description (incidence and severity):

There was no test item effect on mortality or survival of the pups (F1 generation).

Description (incidence and severity):

There were apparent test item related differences in the offspring body weights or body weight gains in the High dose group (450 mg/kg bw/day) when compared to the control values .
Reduced body weight compared to Control was recorded on PND 13 in the High dose groups (by 9.6%, statistically significant at p<0.05); reduced body weight gain was also recorded in the period of PND 4-13 (by 11.4%, statistically significant at p<0.01) and PND 0-13 (by 11.2%, statistically significant at p<0.05). These changes were considered as a test item related effect. Although the High dose males had statistically significant reductions and females had no significantly lower food intake and body weight, High dose females did have some trends indicating a food intake effect and they did show gastric irritation and significant signs of non-specific stress (based on thymus histopathology). The High dose pup weights were only slightly below the range of the contemporaneous historical control range, indicating a test item related effect, but it was considered to be related to maternal toxicity; the difference was not considered to be an adverse effect of treatment on pup development.

Description (incidence and severity):

No test item effect was observed on anogenital distance during the study.

Description (incidence and severity):

No test item effect was observed on nipple retention during the study.

Description (incidence and severity):

No test item-related changes were observed in macroscopically examined pups.

-Mortality and clinical observations:
There was no test item effect on mortality or survival of the pups (F1 generation).
The number of viable pups on PND0, PND4 and PND13 as well as pup survival indices on PND0, PND4 and PND13 were comparable to control values in each test item treated group.
There were no significant differences or effects that could be ascribed to the test item treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any dose groups.
Evidence of suckling was recorded for all live born pups in the study.
Based on the external evaluation, most of the pups were normal, clinical signs or abnormalities were recorded for a Control litter (#1510), where fur thin was recorded for several pups on PND12 and PND13. These events were considered as minor, incidental findings, not related to the test item treatment.
-Body weight and body weight gain:
There were apparent test item related differences in the offspring body weights or body weight gains in the High dose group (450 mg/kg bw/day) when compared to the control values.
Reduced body weight compared to Control was recorded on PND 13 in the High dose groups (by 9.6%, statistically significant at p<0.05); reduced body weight gain was also recorded in the period of PND 4-13 (by 11.4%, statistically significant at p<0.01) and PND 0-13 (by 11.2%, statistically significant at p<0.05). These changes were considered as a test item related effect. Although the High dose males had statistically significant reductions and females had no significantly lower food intake and body weight, High dose females did have some trends indicating a food intake effect and they did show gastric irritation and significant signs of non-specific stress (based on thymus histopathology). The High dose pup weights were only slightly below the range of the contemporaneous historical control range, indicating a test item related effect, but it was considered to be related to maternal toxicity; the difference was not considered to be an adverse effect of treatment on pup development.
-Anogenital distance, nipple retention:
No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when litter mean values were compared to control. No nipples/areolae were present in any of the male pups on PND13.
-Thyroid hormone analysis:
In the PND13 pups, there were no statistically significant or biologically relevant thyroid hormone concentration levels recorded in the test groups when compared to the control.
The thyroid gland weights of the PND13 pups were also comparable with the control value in all test item treated groups.
-Pathology:
TERMINAL / F1 Generation (PND13)
Macroscopic Findings
No test item-related changes were observed in macroscopically examined pups.
Microscopic Findings
No histopathological examination was performed on pups (F1 generation).

Key result

Dose descriptor:

NOAEL

Remarks:

pups' (F1 generation) development and survival

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No other findings

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Based on the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered:
The NOAEL for reproductive effects of the parental generation: 450 mg/kg bw/day (based on the lack of relevant findings).
The NOAEL for pups’ (F1 generation) development and survival: 450 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 450 mg/kg bw/day; although pup growth and body weight were lower than the concurrent control, but they were considered to be related to maternal toxicity).

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of TENSOMILD H026 (containing Disodium lauryl sulfosuccinate (EC 939-638-8) as active ingredient) 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 based on the results of the Dose Range Finding (DRF) study of the test item performed at the Test Facility (Study code: 20/125-220PE) and information from an OECD No. 422 study of a similar monoester compound (available at Sponsor). Based on those results, 450 mg/kg bw/day was selected as the High dose of this study. The experimental design is shown in the table below:

Group Number	Group designation	Dose level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Animal numbers
					Males	Females
1	Control	0	0	5	1001-1012	1501-1512
2	Low dose	60	12		2001-2012	2501-2512
3	Mid dose	150	30		3001-3012	3501-3512
4	High dose	450	90		4001-4012	4501-4512

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 (Day 28 for males, PPD (Post-partum Day) 14 for females), 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 PND (Post-natal Day) 13 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, the retained reproductive organs of all Control and High dose animals, stomach and thymus from all animals, the found dead animal or all organs with relevant macroscopic changes, and the male / female mating pair where no liveborn pups were achieved.

RESULTS

Dosing formulations were analysed for concentration and/or homogeneity on three occasions during the study. Overall, the formulations were considered adequate for the study.

In summary, under the conditions of this study the daily administration of TENSOMILD H026 (containing Disodium lauryl sulfosuccinate (EC 939-638-8) as active ingredient) by oral gavage to Wistar rats at dose levels of 60, 150 or 450 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or clinical signs.

Test item related adverse effect was observed on body weight parameters and food consumption in High dose (450 mg/kg bw/day) males.

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

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

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation.

The number of implantation or liveborn pups, as well as the pre-natal, post-natal or total mortality values were comparable with the control values in all test item treated dose groups. No test item related macroscopic findings were recorded for F1 pups at necropsy.

There were test item-related differences on the offspring body weights or body weight gains in the High dose group (450 mg/kg bw/day) when compared to the study control values, and the High dose pup weights and weight gain were slightly below the contemporaneous historical control range. However, those differences were considered to be related to maternal toxicity (secondary effect), they were not considered as a test item related direct adverse effect.

Increased liver weights in High dose females (450 mg/kg bw/day) were not confirmed at histopathology, but adaptive hypertrophy of 11-14% as seen in this study is often not visible at histopathology; the organ weight difference was considered as non-adverse.

Test item-related changes were observed in the non-glandular stomach in both sexes of the Mid and High dose groups (150 and 450 mg/kg bw/day, respectively) with a dose dependent trend in incidence and severity, indicating local irritation by the test item; males were more affected.

The changes in the non-glandular stomach observed in this study were considered due to the irritant nature of the test item and is considered as a local effect rather than systemic toxicity of the test item. Such changes are commonly associated with lower food intake, lower weight gain (often transient, in the first week of the study) and non-specific stress effects in rats.

No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis.

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

The NOAEL for systemic toxicity of the parental generation: 150 mg/kg bw/day (based on body weight and food consumption effects in males at 450 mg/kg bw/day).

The NOAEL for local toxicity of the parental generation: 60 mg/kg bw/day (based on the local irritation in the stomach in both sexes in the 150 and 450 mg/kg bw/day dose groups).

The NOAEL for reproductive effects of the parental generation: 450 mg/kg bw/day (based on the lack of relevant findings).

The NOAEL for pups’ (F1 generation) development and survival: 450 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 450 mg/kg bw/day; although pup growth and body weight were lower than the concurrent control, but they were considered to be related to maternal toxicity).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive screening

A key subacute OECD No. 422 study was conducted with the registered substance in Wistar rats (12/sex/group) by oral gavage at 0 (distilled water), 60, 150 and 450 mg/kg bw/day (Hargitai, 2022). The systemic toxicity parameters are reported under the Section 7.5. 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 (Day 28 for males, PPD (Post-partum Day) 14 for females), 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 PND (Post-natal Day) 13 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, the retained reproductive organs of all Control and High dose animals, stomach and thymus from all animals, the found dead animal or all organs with relevant macroscopic changes, and the male / female mating pair where no liveborn pups were achieved.

There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation. The number of implantation or liveborn pups, as well as the pre-natal, post-natal or total mortality values were comparable with the control values in all test item treated dose groups. No test item related macroscopic findings were recorded for F1 pups at necropsy. There were test item-related differences on the offspring body weights or body weight gains in the High dose group (450 mg/kg bw/day) when compared to the study control values, and the High dose pup weights and weight gain were slightly outside the contemporaneous historical control range. However, as those differences were related to maternal toxicity (secondary effect), they were not considered as a test item related direct adverse effect. No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis. Based on the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were considered: The NOAEL for reproductive effects of the parental generation: 450 mg/kg bw/day (based on the lack of relevant findings). The NOAEL for pups’ (F1 generation) development and survival: 450 mg/kg bw/day (based on the lack of evidence of a direct effect on the pups at 450 mg/kg bw/day; although pup growth and body weight were lower than the concurrent control, but they were most probably related to maternal toxicity).

 Conclusion

Reproductive toxicity screening was performed with the registered substance in Wistar rats by oral gavage at 0 (distilled water), 60, 150 and 450 mg/kg bw/day in combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD No. 422). The NOAEL for reproductive effects of the parental generation was 450 mg/kg bw/day and the NOAEL for pups’ (F1 generation) development and survival was 450 mg/kg bw/day.

 

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study was not available for the registered substance, however read across data were available from source substances CAS No. 37294-49-8 (Disodium C-isodecyl sulphonatosuccinate) and (CAS 577-11-7) (Docusate sodium). Further prenatal developmental toxicity  testing is  planned and will be updated later when results are available (currently waived in the dossier).

A combined reproduction-teratogenicity study with read across substance CAS No. 37294-49-8 (Disodium C-isodecyl sulphonatosuccinate) in 2 generations resulted in a NOAEL for embryotoxicity of 750 mg/kg (1% in the diet) and a NOEL for teratogenicity of 3000 mg/kg (4% in the diet).

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL corresponding to 1074 mg/kg bw,  whereas at 2% in the diet visceral and skeletal anomalies were observed, which was considered  secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at  subtoxic and toxic dose levels, where the same could be observed. Based on the absence of developmental findings in the screening study and teratogenicity study, no further testing is needed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

See attached read-across justification

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Among rats given dietary levels of 2.0% DSS, there were significant depressions in maternal weight-gains.
Rats fed diets containing 1.0% level of DSS showed no significant maternal effects on the various parameters.

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

Rats fed diets containing 1.0% level of DSS) showed no significant maternal effects on the various parameters. Among rats given dietary levels of 2.0% DSS, there were significant depressions in maternal weight gains.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

In the 2.0% DSS group 1 pregnancy with total resorptions was observed (No statistical significance). No pregnancy with total resorptions was observed in the control or 1.0% DSS group.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Among rats given dietary levels of 2.0% DSS, there were significant increases in the number of resorptions of 13.7% as compared to the control frequency of 5.6%.

Dead fetuses:

no effects observed

Description (incidence and severity):

0.5% occurrence of dead fetuses was seen in the 2.0% DSS group versus 0.7% in the control group. No dead fetuses were observed in the 1.0% DSS group.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined

Changes in number of pregnant:

not examined

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: 2.0% in the diet

Key result

Dose descriptor:

NOAEC

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

in the diet

Basis for effect level:

body weight and weight gain

early or late resorptions

Key result

Dose descriptor:

NOAEL

Effect level:

1 074 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

body weight and weight gain

early or late resorptions

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There is no postnatal evaluation in an OECD 414 study.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There is no significant reduction in viable fetuses in the dosed animals animals compared to control animals.

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

Description (incidence and severity):

There is no postnatal evaluation in an OECD 414 study.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Near toxic or toxic dietary levels of 2.0% DSS produced significant incidences of gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to none in the controls. These abnormalities consisted of cranial buble, exencephaly, spina bifida (not significant), microphtalmia or anophtalmia (not significant).

Skeletal malformations:

no effects observed

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

The visceral observations confirmed the significance of the exencephalous characteristics and anophtalmia for the group given dietary levels of 2.0% DSS.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In the 2.0% DSS group, skeletal observations revealed a significant incidence of variations including incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs.

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
See Table 1-4.

Key result

Dose descriptor:

NOAEC

Effect level:

1 other: %

Based on:

act. ingr.

Remarks:

diet

Sex:

male/female

Basis for effect level:

external malformations

visceral malformations

other:

Remarks on result:

other: secondary to high maternally toxic dose

Key result

Dose descriptor:

NOAEL

Effect level:

1 074 mg/kg bw/day

Based on:

act. ingr.

Remarks:

diet

Sex:

male/female

Basis for effect level:

external malformations

visceral malformations

other: skeletal variations

Abnormalities:

effects observed, treatment-related

Localisation:

external: cranium

skeletal: skull

skeletal: rib

visceral/soft tissue: central nervous system

visceral/soft tissue: eye

Description (incidence and severity):

only at 2.0% dietary level.

Developmental effects observed:

yes

Lowest effective dose / conc.:

2 other: %

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Table 1. Maternal and fetal results of pregnant rats given various amounts if DSS in their diets during  gestational days 6 through 15.

Parameter	Control	1.0% DSS	2.0% DSS
Maternal	Group  (I-A)	(II-A)	(II-B)
No. of pregnant rats	43	22	20
No. of pregnancies with total resorptions	0	0	1
No. of pregnancies with viable fetuses	43	22	19
Average weight gain of dams with viable fetuses(g):
Days 6 to 15	78	86	52*
Days 15 to 21	66	67	77
Average, apparent food intake of dams with viable fetuses (g/rat/day):
Days 6 to 15	22.5	24.8	21.4
Days 15 to 21	28.6	32.1	33.4
Calculated compound consumed (mg/kg/day)	--	1074	1988
Litters
Total number of: implantations	411	203	219
Resorptions (% occurence)	23 (5.6)	8 (3.9)	30*^a (13.7)
Dead fetuses (% occurrence)	3 (0.7)	0	1 (0.5)
Viable fetuses (% occurrence)	385 (93.7)	195 (96.1)	188 (85.5)
Fetal weight (g)	4.6	5.2	4.7
Litters size (viable fetuses)	8.9	8.9	9.9
External major malformations^1: No. of litters affected (% occurrence)	0	0	5* (25.0)
No. of fetuses affected (% occurrence)	0	0	36*^a (20.2)

* Significantly different from control (p< 0.05)

^a Significance by Chi-square, but not Mann-Whitney U test

¹ Primarily, exencephaly varying degrees and associated anomalies (See Table 2)

    

Table 2. Morphological observations of fetuses delivered from rats given DSS in their diets on gestational days 6 through 15.

Morphology	Control	1.0% DSS	2.0% DSS
External observations^1:	Group (I-A)	(II-A)	(II-B)
Total number examined	388^a	195	189
Major anomalies:   Adactyly	0	0	0
Hemimelia	0	0	0
Schistocelia	0	0	2
Dome shaped head	0	0	0
Cranial bubble (1-2mm)	0	0	9*
Exencephaly	0	0	18*
Exencephaly (cleft condition)	0	0	7*
Anencephaly	0	0	0
Spina bifida	0	0	6
Macroglossia	0	0	0
Micro- or anophtalmia	0	0	3
Defects:   Hematoma (subcutaneous)	2	0	0
Edamatous abdomen	0	0	0
Tail short & curled	0	0	0
Abducted fifth digit, left    Rear foot	0	0	1

¹ Fetuses may have more than one defect

^a Fifty-four fetuses examined grossly only. (Shipment c valid as controls only)

      *Significantly different from control (p< 0.05) by Chi-square only

 

Table 3. Visceral observations of fetuses delivered from rats given DSS in their diets on gestation days  6 through 15.

Visceral observations	Dose:      Control	1.0 % DSS	2.0% DSS
	Groups:       (I-A)	(II-A)	(II-B)
Total number of fetuses examined	165^a	98	91
Defects^1:   Exencephalous   characteristics	0	0	11*
Dilated lateral ventricles	1	3	5
Microphtalmia	0	1	0
Anolphtalmia	0	0	23*
Retinal foldings	0	0	0
Anotia or microtia	0	0	0
Cleft palate	0	0	1
Situs transversus – aorta, esophagus   & stomach	1	0	0
Intestinal agenesis	0	0	0
Arch of aorta absent or right sided	0	0	0
Diaphragmic hernia	0	0	1
Dilated renal pelves	2	0	3
Ectopic kidneys(s) &/or variation in size	1	0	0
Renal agenesis	0	0	2
Dilated ureters	6	0	3
Adrenal agenesis	0	0	1
Testes – ectopic or enlarged	1	0	1
Hermaphroditism	0	0	3

¹Fetuses may have more than one defect

^aExcludes 1 fetus lost

*Significantly different from control (p<0.05) by Chi-square only

Table 4. Skeletal observations of fetuses delivered from rats given DSS in their diets on gestation days  6 through 15.

 

Skeletal observations	Dose:      Control	1.0 % DSS	2.0% DSS
	Group  (I-A)	(II-A)	(II-B)
Total number of fetuses examined	167^a	97	98
Defects^1:   Cranial bones,   incomplete to lack of ossification :    Nasal	0	0	4
Frontal	1	0	20*
Parietal	1	1	19*
Interparietal	1	2	18*
Supraoccipital	0	0	15*
Exoccipital	0	0	2
Atlas	0	0	1
Zygomatic	0	0	1
Premaxilla	0	0	1
Tympanic bullae	0	0	5
Mandibles	0	0	1
Hyoid	0	0	3
Eye orbit, reduction	0	0	0
Exoccipital, fused to atlas	0	0	0
Vertebrla column, curved &/or open	0	0	5
Vertebrae:
misshapened &/or retarded     development	0	0	5
thoracic, bipartite centra	2	1	5
lumbar, bipartite centra	0	0	2
Sternebrae:
fused	0	0	0
hypoplastic to absent	0	0	1
one or two absent	1	0	0
staircase	0	0	3
bipartite	0	0	2
Rib(s):
accesory	6	5	5
Absent or less developed	0	0	7*
wavy	2	2	0
fused	0	0	2
Pelvic, hypoplastic to absent	0	0	0
Brachydactyly	0	0	0
Syndactyly	0	0	0
Adactyly	0	0	0
Hemimelia & small scapula	0	0	0

¹Fetuses may have more than one defect

^aExcludes 1 fetus destroyed during cleaning process

*Significantly different from control (p<0.05) by Chi-square only

 

Conclusions:

Subtoxic dietary levels of 1.0% read-across test item docusate sodium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% DSS produced significant incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. Interpretation of the results of the present experiments, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.

Executive summary:

Prenatal developmental toxicity was studied in rats dosed from day 6 to day 15 of gestation by dietary administration of read-across test item docusate sodium at dose levels of 1.0 and 2.0 % in the diet. Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 2.0% docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared the controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophtalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophtalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. There were significant depressions in maternal weight gains in the 2.0% DSS-group. Interpretation of the results of the present experiment, in which only maternally toxic dose levels induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants.

The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with 1074 mg/kg body weight, as calculated in the study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 074 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimish 2

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A prenatal developmental toxicity study was not available for the registered substance, however data were available from read-across substances CAS No. 37294-49-8 (Disodium C-isodecyl sulphonatosuccinate) and CAS 577-11-7 (Docusate sodium, a Diester).  Further developmental toxicity  testing is  planned and will be updated later when results are available (currently waived in the dossier).

Weight of evidence for absence of teratogenicity and developmental toxicity was available from following studies:

- Combined reproduction-teratogenicity study with read across test item CAS No. 37294-49-8 (Disodium C-isodecyl sulphonatosuccinate) in 2 generations (Tegeris and Underwood, 1975c). The test item containing +-50% active ingredient was fed to Charles River CD Sprague-Dawley rats at 1% and 4% in the diet while the control group received normal diet. The original females were allowed to deliver their first 2 litters (F1a & F1b), while the third litters were used for teratological evaluation and partial histology on 5 pairs per group (F1c). Females were allowed to rest 20 days between weaning and their next breeding.

All litters were standardized to 8 newborn to equalize the maternal stress.

a. F1a pups were examined to calculate Fertility Index (FI), Viability Index (VI) and Lactation Index (LI); they were discarded at weaning.

b. F1b pups were examined to calculate Fertility Index (FI), Viability Index (VI) and Lactation Index (LI), with  part of them that were studied postmortem (autopsy and 5 pairs per group for histology) and the other part were selected for second generation breeding, leading to F2a (discarded at weaning) and F2b (teratological examination and partial histology on 5 pairs per group).

c.F1c were used for teratological evaluation an d partial histology on 5 pairs per group.

Although data suggest that the test material under the conditions of this experiment is not teratogenic in the rat it does, however, appear to depress the rate of body weight gain in the pups at 4% in the diet. The number of live born pups , and related to this Fertility Index (FI), Viability Index (VI) and Lactation Index (LI) also decreased at 4%. There were no histological effects on the gonads. NOEL for teratogenicity is 3000 mg/kg (4% in the diet); NOAEL for embryotoxicity is 750 mg/kg (1% in the diet).

In conclusion, a combined reproduction-teratogenicity study with read across substance CAS No. 37294 -49 -8 did not indicate potential for developmental toxicity.

Teratogenicity testing

Further data on prenatal developmental toxicity were available from read across substance Docusate sodium (CAS No. 577-11-7). Justification for read across with the category of Di-ester sulphosuccinates is documented in a separate document attached in Section 13.

-A key study for prenatal developmental toxicity was performed in rats dosed from day 6-15 of gestation with read across substance Docusate sodium dosed at dietary dose levels of 1.0 and 2.0 % in the diet (Roell et al., 1976). The study was conducted according to OECD 414 guideline, and was considered to be reliable, adequate and relevant. Subtoxic dietary levels of 1.0% showed no adverse effects on the various maternal or fetal parameters. Toxic dietary levels of 2.0% Docusate sodium produced significant depressions in maternal weight-gains and increased incidences of resorptions (13.7%) and gross abnormalities either among litters (25.0%) or fetal populations (20.2%) as compared to controls. These abnormalities consisted primarily of exencephaly of varying degrees with, at times, spina bifida, anophthalmia and associated skeletal defects. The visceral observations confirmed the significance of the exencephalous characteristics and anophthalmia for the group given dietary levels of 2.0%. In this group, skeletal observations revealed a significant incidence of incomplete ossification to absence of the various cranial bones, a curved or open vertebral column, and a variety of defects of the vertebrae and ribs. Interpretation of the results of the present experiment, in which only maternally toxic doses induce teratogenicity, indicates no real hazard with the recommended human use of these surfactants. The concentration of 1% in the diet is considered as maternal and developmental NOAEL. This dose level corresponded with a test article intake of 1074 mg/kg body weight, as calculated in the study.

- As supporting information, prenatal developmental toxicity was also studied in rats by dietary administration of Docusate 'calcium' (DCS) at dose levels of 0.5, 1.0, 1.5 and 2.0 % in the diet as well as by oral gavage at 250, 500, 750 and 1000 mg/kg bw (Roell et all., 1976). Subtoxic dietary levels of 0.5 and 1.0% Docusate calcium ingested on gestational days 6 through 15 showed no adverse effects on the various maternal or fetal parameters. Near toxic or toxic dietary levels of 1.5 and 2.0% DCS produced significant incidences of resorptions and gross abnormalities consisting primarily of exencephaly of varying degrees with spina bifida, anophthalmia and associated skeletal defects. However, dietary levels of 2% of DCS fed to pregnant rats for 3 days (days 6-8, 8-10 or 10-12) did not produce teratogenic response. Also, DCS given to pregnant rats by oral intubation at maternally subtoxic doses (250-750 mg/kg) and a slightly toxic dose (1000 mg/kg) did not lead to malformations, however the incidence of resorptions was increased at the 2 toxic doses. Likewise doses of 500 and 750 mg/kg given by gavage from day 6-15 produced an increase in resorptions at the highest dose without a teratogenic effect. Since only maternally toxic doses fed on gestational day 6-15 produced embryotoxic and teratogenic effects, it is concluded that no real hazard exists. 

In conclusion, both a prenatal developmental toxicity with read across substance Docusate sodium and with read across substance Docusate calcium were negative for teratogenicity at non-toxic dose levels.

 

Conclusion

Combined reproduction-teratogenicity study with read across test item Disodium C-isodecyl sulphonatosuccinate (CAS No. 37294-49-8) in 2 generations resulted in NOAEL for embryotoxicity  of 750  mg/kg (1% in the diet) and a NOEL for teratogenicity of 3000 mg/kg (4% in the diet).

Prenatal developmental toxicity was tested by dietary administration of read across substance Docusate sodium (CAS 577-11-7) in rats from day 6 to 15 of gestation. 1% in the diet was a maternal and developmental NOAEL corresponding to 1074 mg/kg bw, whereas at 2% in the diet visceral and skeletal anomalies were observed, which were considered secondary to maternal toxicity. This was confirmed in a similar study with Docusate calcium given at subtoxic and toxic dose levels, where the same could be observed.

Justification for classification or non-classification

Based on these results and according to CLP (No. 1272/2008 of 16 December 2008), the test substance does not have to be classified and has no obligatory labelling requirement for reproductive and developmental toxicity.