Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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:
This study was performed in full compliance with:
OECD Guidelines for the Testing of Chemicals, No. 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
adopted 29 July 2016
EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test, July 2000
GLP compliance:
yes
Specific details on test material used for the study:
Characteristics of test item

Test item: 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide
CAS: 201419-80-9
Lot number: AZ08AVL1
Active component: 99.9 %
Appearance: Crystalline solid, white
Expiration date: September 21, 2022
Storage conditions: At room temperature, protected from humidity, well-closed container
Species:
rat
Strain:
Wistar
Remarks:
Rat, Han:WIST of Wistar origin
Details on species / strain selection:
Reason for selection of species

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal husbandry

Animal room no.: 17
Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Mated females: individually
Males after mating: 2 animals / cage
Cage type: Type III polypropylene/polycarbonate;
Size: 22 x 32 x 19 cm (width x length x height)
Bedding: Certified laboratory wood bedding (SAFE 3/4-S- FASERN and SAFE 3/4 S; both produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany; see Appendix 22).
The bedding is suitable as nesting material. Details of quality of bedding material were reported.
The cages and bedding were changed once or twice a week.
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: Above 10 air-exchanges/ hour by a central air-condition system.
Environmental conditions were maintained by an air-conditioning system. Temperature and relative humidity were verified and recorded daily during the study.

3.4.4 Food and water supply

Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum and tap water from municipal supply, as for human consumption, from 500 mL bottles ad libitum.
Food was changed at weekly intervals. Fresh drinking water was given daily.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Contents of the standard diet for rats and mice guaranteed by the supplier are presented in Appendix 20.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 174. Budapest, H-1138 Hungary). The quality control results are available at Toxi-Coop Zrt.’s archives (see Appendix 21).

Route of administration:
oral: gavage
Details on route of administration:
Route of administration and reason for the selection

The test item was administered orally via gavage. The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.
Vehicle:
vegetable oil
Remarks:
Vehicle Name: Sunflower oil (Helianthi annui oleum raffinatum) Batch number 1: 8007816003 Expiry date 1: August 31, 2022 Batch number 2: 8007907002 Expiry date 2: October 31, 2022 Manufacturer: Magilab Kft. Király utca 12. H-1061 Budapest Hungary
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration check of the formulated test item

Analytical control of dosing formulations (control of concentration) was performed in the Analytical Laboratory of Test Facility twice during the study.

Five aliquots of 10 mL of each formulation and five aliquots of control substance (vehicle) were taken two times and were analyzed.
Date of sampling: December 08, 2021 and January 26, 2022
Date of analysis: December 10, 2021 and January 27, 2022
Concentration of the test item in the dosing formulations varied between the range of 91.4 % and 106.1 % in comparison to the nominal values. Results and details of analysis are attached to the Report (Appendix 18.2).
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.
The recovery of the test item from the vehicle was within the acceptance criteria (95 and 105 % relative to nominal concentrations) at ca. 1 mg/mL and at ca. 200 mg/mL.
2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide proved to be stable in sunflower oil at the intended concentrations at room temperature for one day and at 5±3 oC for three days.
A separate analytical report (Study no. 993-100-5837) provided these data.
Duration of treatment / exposure:
42 days at male rats
42, 51, 65 days at female rats

Duration of the experimental period

The experimental period involved 20 days of acclimatization (including 14 days for examination of estrous cycle) and 42 (male), 42, 51-65 (female) days treatment/ observation period (depending on the effectiveness of mating) and necropsy days.
The day of first treatment is considered as Day 0 of examination.
Frequency of treatment:
daily for 7 days / week
Dose / conc.:
1.8 mg/kg bw/day (actual dose received)
Dose / conc.:
1.3 mg/kg bw/day (actual dose received)
Dose / conc.:
0.9 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
No. of animals per sex per dose:
12 female and 12 males
Control animals:
yes, concurrent vehicle
Details on study design:
4.1 Selection of animals

48 male and 48 female healthy rats were involved in the study. Animals were selected for this study on the basis of adequate body weight, a body weight within ± 20% of the mean within a sex and free from clinical signs of disease or injury. Animals that failed to exhibit typical
4-5 days cycles were not included in the study if it was feasible.
Selected rats were distributed by randomization according to stratification by body weight so that there was no statistically significant difference among group body weight means within a sex.

Study design

a) Males
A PM M ↓
Acclimatization period
20 days Pre-mating period
14 days Mating
period
1-16 days Post mating period
12-27 days FOB on Day 41, ††
Blood sampling
Necropsy
Organ weighing
on Day 42

b) Females
A PM M G ↓ PP/PN ↓
Acclimatization period
20 days† Pre-mating period
14 days Mating
period
1-16 days Gestation period
22-23 days Delivery Lactation
period
13-16 days FOB on Day 54, ††
Blood sampling, Necropsy, Organ weighing Day 54, ††
Necropsy on
Days 51, 54, 65
Remark: † = Pre-treatment examination of estrous cycle included
†† = For animals selected for toxicity examinations.

Dosing of both sexes begun after 20 days acclimatization – including 14 days pre-treatment estrous cycle examination – and was continued up to and including the day before the necropsy. Rats of this strain reach full sexual maturity at the age of 10 weeks. The mating phase started after 14-days treatment (pre-mating) period.
The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time (±2 hours). Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Male animals were dosed for 42 days (14 days pre-mating and 1-16 days mating, plus
12-27 days of post-mating period; until the necessary number of pregnant female animals was evident) then they were sacrificed on Day 42.

Positive control:
no
Observations and examinations performed and frequency:
4.4 Observations

4.4.1 Estrous cycle

Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization as it was possible.
Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (pre-mating period) and during the mating period until evidence of mating.
Vaginal smears were prepared and evaluated in each female animal on the day of necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

4.4.2 Mortality

Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

4.4.3 Clinical observations

General clinical observations were made on parental animals once a day, after the administration at approximately the same time.
More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling.
Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g., auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week (male animals on Day 41; female animals on Day 54). General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

4.4.4 Body weight

The body weight of all parental animals was determined with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated. Body weight was measured on the day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated according to measurement days and for the study overall (latter named also as summarized body weight gain).

4.4.5 Food consumption measurement

The food consumption was determined weekly by weighing the given and non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 0, 7, 13 and post-mating days 20, 27, 34 and 41 for male animals, pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

4.4.6 Examination of placental sign

All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13.

If the test was negative on day 13, the examination was repeated on day 14 of gestation. Examination of placental signs was only for checking the pregnancy and data were not reported.

4.4.7 Observation of the delivery process

Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestational day 21 onwards. All observations and any evidence of abnormal deliveries were considered and recorded. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if it was feasible. Extra pups were eliminated by a random selection. Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

4.4.8 Observation of the offspring

Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition was complete, post-natal day 0) and on post-natal day 13.
Litter weight were determined with an accuracy of 0.1 g. The litter weight was calculated by the individual weight of pups on post-natal day 4.
Any abnormal behavior of the offspring was recorded.
All litters were checked and recorded daily for the number of viable and dead pups.
The anogenital distance of each pup was determined on post-natal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on post-natal day 4.
The number of nipples/areolae in male pups was counted on post-natal day 13.
Dead or stillborn offspring were subjected to necropsy by macroscopic examination on the day when they were found dead. On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn; negative lung flotation test) from pups died after the birth (dead pups; positive lung flotation test).

4.4.9 Clinical pathology

Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e., on the day of necropsy).
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran CP® anesthesia.

Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry and determination of thyroid hormones (FT3, FT4 and TSH).

4.4.9.1 Hematology

Blood samples for hematology measurements were collected in tubes containing K3EDTA (spray-dried; MiniCollect® 0.5 mL, manufactured by Greiner Bio-One International AG, Kremsmünster, Austria) and tubes were filled up to the final volume (marked on the tubes) and analyzed.
The following parameters were measured in all selected animals by Siemens ADVIA120:

Table 4: Hematology parameters examined
PARAMETERS UNIT METHODS
WBC
White Blood Cell (leukocyte) count 109/L (G/L) Flow cytometry method
RBC
Red Blood Cell (erythrocyte) count 1012/L (T/L) Flow cytometry method
HGB
Hemoglobin concentration g/L Cyanide-colorimetric hemoglobin method
HCT
Hematocrit (relative volume of erythrocytes) L/L Computed by equipment
MCV
Mean Corpuscular (erythrocyte) Volume fL Flow cytometry method
MCH
Mean Corpuscular (erythrocyte) Hemoglobin pg Computed by equipment
MCHC
Mean Corpuscular (erythrocyte) Hemoglobin Concentration g/L Computed by equipment
PLT
Platelet (thrombocyte) count 109/L (G/L) Flow cytometry method
RET
Reticulocytes, % Flow cytometry method
Differential† white blood cell count %, 109/L (G/L) Peroxidase and basophil/ lobularity method
† Notes: NEU: Neutrophil (%, G/L); LYM: Lymphocyte (%, G/L); EOS: Eosinophil; (%, G/L);
MONO: Monocyte (%, G/L); BASO: Basophil (%, G/L)

4.4.9.2 Blood coagulation

Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation 3.2 % (MiniCollect® 1 mL; manufactured by Greiner Bio-One International AG, Kremsmünster, Austria). Tubes were filled up to the final volume (marked on the tubes).
Blood was centrifuged at 2500 rpm for 15 minutes within 20-30 minutes after the sampling. Supernatant plasma samples were measured.
The following blood coagulation parameters were measured in all selected animals by Sysmex CA-1500:


Table 5: Blood coagulation parameters examined
PARAMETERS UNIT METHODS
APTT
Activated partial Thromboplastin Time sec Optical
PT
Prothrombin Time sec Optical

4.4.9.3 Clinical chemistry

Blood samples collected for clinical chemistry measurements were drawn in tubes Vacuette 2.5 mL Z Serum Sep C/A (no anticoagulant; manufactured by Greiner Bio-One International AG, Kremsmünster, Austria). At least 1.0 mL blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. Serum samples were frozen between minus 15 and minus 30 oC and measured.
The following parameters were measured in all selected animals by Cobas C311:

Table 6: Clinical chemistry parameters examined
PARAMETERS UNIT METHODS
ALT
Alanine Aminotransferase activity U/L IFCC recommended
(with P-5’-P), 3-reagent system
AST
Aspartate Aminotransferase activity U/L IFCC recommended
(with P-5’-P), 3-reagent system
TBIL
Total Bilirubin concentration mol/L Colorimetric diazo method (NBD: p-nitrobenzene-diazonium)
CREA
Creatinine concentration mol/L Enzymatic method
UREA
Urea concentration mmol/L Urease-GLDH method
GLUC
Glucose concentration mmol/L Hexokinase method
CHOL
Cholesterol concentration mmol/L Enzymatic CHOD-POD method
Na+
Sodium concentration mmol/L Potentiometric test
(Indirect ISE)
K+
Potassium concentration mmol/L Potentiometric test
(Indirect ISE)
ALB
Albumin concentration g/L Colorimetric - BCG
(Bromocresol green) - metod
TPROT
Total Protein concentration g/L Colorimetric – Biuret - method

4.4.9.4 Determination of serum thyroid hormone

Blood samples were collected from animals as follows:
- from 2-6 pups per litter on post-natal day 4, as the number of pups allowed
- from all dams and 4-7 pups per litter on post-partum/ post-natal day 13
- from all parent male animals and non-pregnant female animals at termination (Day 42) 
Blood samples for determination of serum levels of thyroid hormones (FT3, FT4 and TSH) were drawn as described at 4.4.9.3 in the morning hours. Each serum sample for thyroid hormones was divided into two aliquots (one for examination of FT3 and FT4, the second one for TSH examination).
All samples were stored under appropriate conditions (between minus 15 and minus 35 °C) until measurement.
Blood samples from the day 13 pups and the adult males were assessed for serum levels of thyroid hormones. Further assessment of FT3, FT4 and TSH in blood samples from the day 4 pups and parental females was not performed as it was considered not relevant (test item related changes were not detected in the thyroid hormones of day 13 pups and the adult male animals or in histopathology of hypothalamic- pituitary-thyroid axis).
The quantitative determination of FT3 and FT4 concentrations in serum samples were performed by electrochemiluminescence immunoassay with an immunochemistry analyzer – COBAS e411 – in the test facility (Toxi-Coop Zrt.).
The serum TSH level was determined by radioimmunoassay method using Berthold Multi Crystal Gamma Counter LBIS 12 detectors including LBIS software, 230W at the Test Site (Izotóp Intézet Kft.).
The following parameters were deetermined:

Table 7: Thyroid hormone parameters examined
PARAMETERS UNIT METHODS
FT3
Free three-iodothyronine ng/dL Electrochemiluminescence immunoassay
FT4
Thyroxine – free Tetra-iodothyronine ng/dL Electrochemiluminescence immunoassay
TSH
Thyroid-stimulating hormone ng/mL Radioimmunoassay




Sacrifice and pathology:
4.4.10.1 Necropsy

Gross necropsy was performed on each animal.
- Parental male animals and non-pregnant female animals: on Day 42.
- Dams not selected for toxicology examinations: on post-partum day 14 or shortly thereafter (Days 51, 54 or 65);
- Dams selected for toxicology examinations: shortly after post-partum day 13 (post-partum days 14-17) on Day 54.
- Offspring: on post-natal day 13 or shortly thereafter.
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution. 
Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter for the intended subsequent histopathological examination. The appearance of the tissues and organs was observed in pups selected for thyroid gland preservation. Thyroid and parathyroid were preserved together with larynx.
Pups euthanized on day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected from each group:

Table 8: List of organs preserved
Adrenal glands
Aorta
Bone with bone marrow and joint (femur)
Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata)
Esophagus
Eyes (lachrymal gland with Harderian glands)
Heart
Kidneys
Large intestines (caecum, colon, rectum)
Liver
Lungs (with main stem bronchi; inflation with fixative and then immersion;)
Lymph nodes (submandibular, mesenteric)
Mammary gland
Muscle (quadriceps)
Pancreas
Pituitary
Salivary glands (submandibular)
Sciatic nerve
Sexual organs (testes, epididymides, prostate seminal vesicle with coagulating gland
ovaries, uterus with cervix and oviduct, vagina)
Skin
Small intestines (duodenum, ileum, jejunum, including Peyer’s patches)
Spinal cord (at three levels: cervical, mid-thoracic and lumbar)
Spleen
Sternum
Stomach
Thymus
Thyroid + parathyroid
Trachea
Urinary bladder
Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.

4.4.10.2 Organ weight

At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Paired organs were weighed together.
In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.
Absolute organ weight was recorded. Relative organ weights (to body and brain weight) were calculated and reported.

4.4.10.3 Histopathology

Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1.8 mg/kg bw/day) groups,
Additionally, kidneys and skin with necropsy findings were processed and evaluated histologically in some parental animals and offspring as follows:
- Kidneys: male animals no. 106, 202, 301, 305, 408, female animals: 228, 326, 421, 425
- Skin: female animal no. 128
- Uterus: female animal no. 224
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Statistics:
Statistical evaluation

The statistical evaluation of appropriate data (marked †above) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.

Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance with respect to the control was detected at the slightly higher mean body weight gain in male animals at 0.9, 1.3 or 1.8 mg/kg bw/day during the last week of the study (between Days 34 and 41). This higher mean body weight gain did not result in significant changes in the mean body weight or in the summarized body weight gain (between Days 0 and 41). Statistical significance was originated the unusually lower mean body weight gain in the control group during the last week.
The mean body weight gain was similar in the control and test item administered female animals during the entire study.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical significance with respect to their control was observed at the slightly lower mean liver weight relative to brain weight in male animals at 1.8 mg/kg bw/day and at the lower mean spleen weights (absolute and relative to brain weight) in the female animals at 0.9 mg/kg bw/day. The weights of liver and spleen (absolute or relative to brain weights) were well within the historical control range and there were no related morphological changes at the histopathology.

Therefore, these slight variations were considered as a toxicologically not relevant effect.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Pyelectasia, hydrometra and alopecia are frequent observations also in untreated experimental rats of this strain with similar age and similar occurrence noted in this study. The incidence of these findings is well within the historical control range. Hydrometra is related to the female sexual cycle.
In the lack of related inflammatory or other pathological alterations, hydrometra and pyelectasia were judged to be toxicologically not relevant.
Necropsy findings in testes in single animal (smaller than normal) was individual alteration occurring commonly in this strain of experimental rats.
Brownish-red colored thymus was likely to be a consequence of the exsanguination procedure (causing circulatory disturbances) at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In one male animal at 1.8 mg/kg bw/day (1/12), decreased intensity of spermatogenesis in the testes and lack of mature spermatozoa were detected in accordance with macroscopic finding (smaller than normal testes).
In the female animals in control (12/12) and 1.8 mg/kg bw/day (12/12, including non-pregnant animals) groups, the ovaries, uterus, cervix, vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was detected in one female animal at 0.9 mg/kg bw/day in accordance with necropsy finding (hydrometra), which is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrous phase – of inner genital organs.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
One or both sided renal pyelectasia was observed in all groups independently from doses as follows:
- Control: 4/6 male
- 0.9 mg/kg bw/day: 1/1 male and 1/1 female
- 1.3 mg/kg bw/day: 2/2 male and 1/1 female
- 1.8 mg/kg bw/day: 2/6 male and 3/7 female
Pyelectasia without degenerative, inflammatory or other histological (fibrotic etc.) lesion is considered as a common finding in laboratory rats without toxicological significance.
Thymic hemorrhage was probably due to the exsanguination procedure in two male animals at 1.8 mg/kg bw/day (2/5).
Atrophy of hair follicles with focal or multifocal alopecia was observed in one female animal (1/6 control). This dermal alteration is a common finding in laboratory rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Dose descriptor:
NOAEL
Effect level:
ca. 1.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Remarks on result:
other: Highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
Remarks on result:
other: At 1.8 mg/kg bw/day, lowered white blood cell and lymphocyte counts (leukocytopenia and lymphocytopenia) were detected.
Critical effects observed:
yes
Lowest effective dose / conc.:
1.8 mg/kg bw/day (actual dose received)
System:
immune system
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the conditions of the present study, 2,4,8,10-Tetraoxa-3,9-
dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide administered at 0.9, 1.3 and 1.8 mg/kg
bw/day by oral gavage did not adversely influence the reproductive performance (gonad
function, mating behavior, conception, parturition) in male or female Han:WIST rats as
far as investigated in this study.
At 1.8 mg/kg bw/day, lowered white blood cell and lymphocyte counts (leukocytopenia
and lymphocytopenia) were detected.
There were no signs of systemic toxicity in male or female animals at 0.9 or 1.3 mg/kg
bw/day.
The development of the F1 offspring was not impaired from birth up to post-natal day
13 as far as investigated in this study after repeated oral administration of dams at 0.9,
1.3 and 1.8 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) was
determined as follows:
NOAEL for systemic toxicity of male rats: 1.3 mg/kg bw/day
NOAEL for systemic toxicity of female rats: 1.8 mg/kg bw/day
NOAEL for reproductive performance of male/ female rats: 1.8 mg/kg bw/day
NOAEL for F1 Offspring: 1.8 mg/kg bw/day
Executive summary:

The purpose of this study was to obtain initial information on the systemically toxic potential
of test item and on the possible effects of the test item on reproduction and development when
repeatedly administered orally (by gavage) to rats at doses of 0.9, 1.3 and 1.8 mg/kg bw/day
compared to control animals according to OECD 422.
As a screening test, it was intended to provide initial information on the possible health hazards
likely to arise from repeated exposure over a relatively limited period of time and on the possible
effects on male and female reproductive performance such as gonadal function, mating
behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from
conception to day 13 post-partum associated with administration of repeated maternal doses.
2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide was administered orally
(by gavage) once daily at 0 (vehicle only), 0.9, 1.3 and 1.8 mg/kg body weight (mg/kg bw/day)
doses to four groups of Han:WIST rats consisting of 12 animals per sex in all groups in
concentrations of 0, 0.18, 0.26 and 0.36 mg/mL corresponding to a 5 mL/kg bw dosing volume.
A group of vehicle (sunflower oil) treated animals served as a control.
The suitability of the chosen vehicle for the test item at the intended concentrations was
analytically verified up front. 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-
tetraoxide proved to be stable in sunflower oil at the intended concentrations at room
temperature for one day and at 5±3 oC for three days.
The concentration of the test item in the dosing formulations administered to the animals was
checked twice during the study. 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5]undecane, 3,3,9,9-
tetraoxide concentrations in the dosing formulations varied within the range of 91.4 % and
106.1 % (in comparison to the nominal values) and confirmed the proper preparation of the
dosing formulations.
All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout
mating. In addition, male and non-pregnant female animals received the test item or vehicle
after mating up to the day before the necropsy (altogether for 42 days). Dams were
additionally exposed through the gestation period and up to lactation days 13-16, i.e., up to
the day before necropsy (altogether for 51, 54 or 65 days).
Observations included mortality, clinical signs, body weight, food consumption, mating,
pregnancy and delivery process, as well as development of offspring. Estrous cycle was monitored
by examining vaginal smears before the treatment for two weeks and for two weeks from the
beginning of the treatment period and during the mating period until evidence of copulation.
Vaginal smears were also prepared and investigated on the day of the necropsy for each dam.
The dams were allowed to litter and rear their offspring up to day 13 post-partum. Litters were
weighed and offspring were observed for possible abnormalities and were euthanized on postnatal day 13 or shortly thereafter.
Blood samples were collected for determination of serum levels of thyroid hormones (FT3, FT4
and TSH) from 2-6 pups per litter (in litters with at least 10 pups) on post-natal day (PND) 4, from
all dams and from 4-7 pups per litter at termination on post-partum/post-natal day 13 and from all
parent male animals and non-pregnant female animals at termination. Serum level of FT3, FT4
and TSH were determined in the samples of parental male animals and PND 13 offspring.


Five dams and their male mating partners were randomly selected from each group to examine
further signs of toxicity such as functional observations, hematology and blood coagulation,
clinical chemistry, gross necropsy, organ weighing and histopathology examinations.
All parental animals were subjected to gross pathology one day after the last treatment. The
body weight, brain weight and weight of the testes and epididymides and prostate and seminal
vesicles with coagulating glands as a whole of adult male animals were determined. In
addition, for five males and females randomly selected from each group, adrenal glands, brain,
heart, kidneys, liver, spleen, thymus and thyroid glands were weighed.
Thyroid glands were preserved from all adult males and females and one male and one female
pup per litter for the intended subsequent histopathological examination.
Histopathology examination was performed on ovaries, uterus, vagina, testes, epididymides,
prostate and seminal vesicles with coagulating gland in all animals (male or female) in the
control and high dose groups.
Full histopathology examinations were performed on the preserved organs and tissues of the
randomly selected animals in the control and high dose groups. In addition, organs showing
macroscopic findings at the necropsy were also processed and examined histologically in
parental animals (kidneys, skin).
The results of this study were summarized as follows:
Mortality
No unscheduled death occurred during the study.
Clinical and functional observation
There were no clinical signs related to the test item in male or female animals at the daily or
detailed weekly clinical observations.
Functional observation battery did not demonstrate any alterations in the behavior or reactions
to different type of stimuli of selected male or female animals in the control, 0.9, 1.3 or
1.8 mg/kg bw/day groups at the end of the treatment period.
Body weight and body weight gain
The body weight development was not influenced in male or female animals at 0.9, 1.3 or
1.8 mg/kg bw/day during the entire treatment period.
Food consumption
The mean daily food consumption was similar in the control and 0.9, 1.3 or 1.8 mg/kg bw/day
dose levels during the entire treatment period (pre-mating and post-mating periods in male
animals; during the pre-mating, gestation and lactation periods in female animals).
Estrous cycle
A test item influence on the estrous cycle was not detected at any dose level (0.9, 1.3 or
1.8 mg/kg bw/day).
Delivery and pregnancy data of female animals
There were no toxicologically relevant differences in the evaluated parameters of delivery
between the control and test item treated groups (0.9, 1.3 or 1.8 mg/kg bw/day).


Reproductive performance
The examined parameters of reproductive performance were not affected by the test item at
0.9, 1.3 or 1.8 mg/kg bw/day in male or female animals.
Hematology and blood coagulation
Significantly lowered mean white blood cell count along with lowered lymphocyte count were
observed in male animals at 1.8 mg/kg bw/day.
Clinical chemistry
Clinical chemistry investigation did not reveal test item related changes in the examined
hematology, blood coagulation or clinical chemistry parameters at 0.9, 1.3 or 1.8 mg/kg
bw/day.
Serum thyroid hormones
The thyroid hormone (FT3, FT4 and TSH) levels were not affected by the test item in parental
male and female animals (0.9, 1.3 and 1.8 mg/kg bw/day) and in offspring sampled on postnatal day 13.
Necropsy
Gross necropsy observations revealed no test item related changes in the organs and tissues
of male or female animals at 0.9, 1.3 or 1.8 mg/kg bw/day
.
Organ weight
There were no adverse test item related alterations in the weights of the examined organs in
male or female animals at 0.9, 1.3 or 1.8 mg/kg bw/day.
Histopathology
There were no toxic or other test item related lesions detectable by histological examination
in the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate
and seminal vesicles with coagulating gland) of male or female animals administered with
1.8 mg/kg bw/day.
Offspring
The offspring’s development was undisturbed at 0.9, 1.3 and 1.8 mg/kg bw/day from birth to
post-natal day 13. No effect on the mortality, clinical signs, body weight development,
anogenital distance (male and female) or nipple retention (male) were detected.


Under the conditions of the present study, 2,4,8,10-Tetraoxa-3,9-
dithiaspiro[5.5]undecane, 3,3,9,9-tetraoxide administered at 0.9, 1.3 and 1.8 mg/kg
bw/day by oral gavage did not adversely influence the reproductive performance (gonad
function, mating behavior, conception, parturition) in male or female Han:WIST rats as
far as investigated in this study.
At 1.8 mg/kg bw/day, lowered white blood cell and lymphocyte counts (leukocytopenia
and lymphocytopenia) were detected.
There were no signs of systemic toxicity in male or female animals at 0.9 or 1.3 mg/kg
bw/day.
The development of the F1 offspring was not impaired from birth up to post-natal day
13 as far as investigated in this study after repeated oral administration of dams at 0.9,
1.3 and 1.8 mg/kg bw/day.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) was
determined as follows:









NOAEL for systemic toxicity of male rats:
NOAEL for systemic toxicity of female rats:
NOAEL for reproductive performance of male/ female rats:
NOAEL for F1 Offspring:
1.3 mg/kg bw/day
1.8 mg/kg bw/day
1.8 mg/kg bw/day
1.8 mg/kg bw/day

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.3 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1
System:
immune system
Organ:
blood

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification