2,4,8,10-tetraoxa-3λ6,9λ6-dithiaspiro[5.5]undecane 3,3,9,9-tetraoxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,4,8,10-tetraoxa-3λ⁶,9λ⁶-dithiaspiro[5.5]undecane-3,3,9,9-tetrone

EC Number:

859-869-7

Cas Number:

201419-80-9

Molecular formula:

C5H8O8S2

IUPAC Name:

2,4,8,10-tetraoxa-3λ⁶,9λ⁶-dithiaspiro[5.5]undecane-3,3,9,9-tetrone

Test material form:

solid: crystalline

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

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Rat, Han:WIST of Wistar origin

Details on species / strain selection:

3.4.2 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:

Species / Strain: Rat, Han:WIST of Wistar origin
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
Number of animals: 48 males, 48 females (nulliparous, non-pregnant females)
Number of animals/group 12 animals/sex/groups (at least 8 pregnant female animals per group are expected);
Number of animals ordered: 55 male and 65 female animals; Randomization reserve was excluded from the study just after randomization.
Age of animals at start of Male animals: 77 – 83 days
the treatment: Female animals: 77 – 83 days
Body weights at start of 327 – 391 g for male animals
the study: 219 – 256 g for female animals
The weight variation did not exceed  20 per cent of the mean weight.
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder (Appendix 19).
Acclimatization time: 20 days

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

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

Details on exposure:

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.

3.5.2 Dose levels

The doses were determined on the basis of the findings obtained from a previously performed oral preliminary toxicity studies with 2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5] undecane, 3,3,9,9-tetraoxide (Study no. 993-400-5839 and 993-400-5839R).
2,4,8,10-Tetraoxa-3,9-dithiaspiro[5.5] undecane, 3,3,9,9-tetraoxide was lethal at 25 mg/kg bw/day (5/5 male and 5/5 female) and at 10 mg/kg bw/day (2/5 male and 1/5 female) and caused severe clinical signs at 3 mg/kg bw/day and transient clinical signs at 2 mg/kg bw/day in male and female rats during the consecutive 14-day oral (by gavage) administration.
Doses of this study were selected with the aim of inducing toxic effects but no death or suffering at the highest dose and a NOAEL at the lowest dose. The mid dose is interpolated geometrically.
A control and three dose groups were involved in the study. Table 3 contains the group number, doses, dosing volume and number of animals.

Table 3: Experimental design
Groups Dose
(mg/kg bw day) Concentration
(mg/mL) Dose volume
(mL/kg bw) Number of animals
Male Female
Group 1 0 0 5 12 12
Group 2 0.9 0.18 5 12 12
Group 3 1.3 0.26 5 12 12
Group 4 1.8 0.36 5 12 12
Animals in Group 1 only received the vehicle.

Details on mating procedure:

Mating procedure

Mating begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred or two weeks elapsed.
One pair (male animal no. 312 and female animal no. 332) at 1.8 mg/kg bw/day failed to mate during 14-day. Therefore, the mating period was prolonged and a new mating partner (no. 311) was cohabited with this female from mating day 15 until successful mating on 16th day.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422). Sperm positive females were caged individually. Mating pairs were clearly identified in the raw data.

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 males
42, 51, 65 at females
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, 7 dasy / week

Details on study schedule:

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.
 
Females were dosed for 14 days pre-mating, through 1-16 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice on Days 51, 54 or 65.
The day of birth (viz. when parturition is complete) was defined as day 0 post-partum. Control animals were handled in an identical manner to the test groups receiving vehicle (5 mL/kg bw).
All animals were subjected to a full detailed gross necropsy. The brain, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole were weighed in all male animals.
Five dams and males these females cohabited with from each group were randomly selected from each group for additional examinations: individual observations in functional observation battery (FOB), clinical pathology examinations, organ weighing and for a full histopathological investigation.
F1 offspring were observed for clinical signs, litter weight, body weight, anogenital distance and nipple retention. Blood samples for serum FT3, FT4 and TSH assessment were pooled from 2-6 pups per litter on post-natal day 4 (in litters with at least 10 pups) and from 4-7 pups per litter on post-natal day 13. Remaining pups were euthanized on post-natal day 13 or shortly thereafter.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

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.
 
Females were dosed for 14 days pre-mating, through 1-16 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice on Days 51, 54 or 65.
The day of birth (viz. when parturition is complete) was defined as day 0 post-partum. Control animals were handled in an identical manner to the test groups receiving vehicle (5 mL/kg bw).
All animals were subjected to a full detailed gross necropsy. The brain, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole were weighed in all male animals.
Five dams and males these females cohabited with from each group were randomly selected from each group for additional examinations: individual observations in functional observation battery (FOB), clinical pathology examinations, organ weighing and for a full histopathological investigation.
F1 offspring were observed for clinical signs, litter weight, body weight, anogenital distance and nipple retention. Blood samples for serum FT3, FT4 and TSH assessment were pooled from 2-6 pups per litter on post-natal day 4 (in litters with at least 10 pups) and from 4-7 pups per litter on post-natal day 13. Remaining pups were euthanized on post-natal day 13 or shortly thereafter.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

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.
 

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

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.

Litter observations:

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).

Postmortem examinations (parental animals):

4.4.10 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.

Postmortem examinations (offspring):

Pups euthanized on day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.

Statistics:

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.

Reproductive indices:

Formulas for calculation of mating and fertility indices

Copulatory index: Measure of animals’ ability to mate
Males ("Number"  "of"  "males"  "with"  "confirmed"  "mating" )/"Total Number of males cohabited"  x "100"
Females ("Number"  "of"  "sperm positive"  "females" )/"Total Number of females cohabited"  x 100

Fertility index: Measure of male’s ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant.
Males ("Number"  "of"  "males"  "impregnating"  a "female" )/"Total Number of males with confirmed mating"  x 100
Females ("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"

5.3 Formulas for calculation of pup mortality and sex ratio indices

Post-implantation mortality (intrauterine mortality)
("Number"  "of"  "implantations - Number of liveborns" )/"Number of implantations"  x 100

Post-natal mortality
("Number"  "of"  "liveborns -Number of live pups on PN13" )/"Number of liveborns"  x 100

Survival Index
("Number"  "of"  "live"  "pups"  "on postnatal day 13" )/"Number of pups born"  x 100

Sex ratio
("Number"  "of"  "pups"  "examined"  - "Number"  "of"  "males " ("females" ))/"Number of pups examined"  x 100

Normalized anogenital distance
"Absolute anogenital distance" /"(Body weight)1/3"

Offspring viability indices:

Formulas for calculation of pup mortality and sex ratio indices

Post-implantation mortality (intrauterine mortality)
("Number"  "of"  "implantations - Number of liveborns" )/"Number of implantations"  x 100

Post-natal mortality
("Number"  "of"  "liveborns -Number of live pups on PN13" )/"Number of liveborns"  x 100

Survival Index
("Number"  "of"  "live"  "pups"  "on postnatal day 13" )/"Number of pups born"  x 100

Sex ratio
("Number"  "of"  "pups"  "examined"  - "Number"  "of"  "males " ("females" ))/"Number of pups examined"  x 100

Normalized anogenital distance
"Absolute anogenital distance" /"(Body weight)1/3"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In two female animals, alopecia was observed as follows:
- 1/12 control on the left side of the abdomen between lactation days 4 and 14
- 1/12 at 1.3 mg/kg bw/day on the lower part of the back from study Day 7 up to and including gestation day 19
Alopecia is a species-specific finding of this strain of rat with similar age.
All other female animals were normal in the control and test item administered groups at the daily and weekly clinical observations during the observation period: pre-mating, mating, gestation and lactation periods, non-pregnant female animals during post-mating period.

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

Description (incidence and severity):

Hematological evaluation revealed changes in white blood cell parameters (lymphocytopenia and leukocytopenia) in male animals at 1.8 mg/kg bw/day. Similar findings were observed in a lesser degree in female animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistical significance with respect to the control was only detected at the slightly lower mean total bilirubin level (TBIL) in male animals at 1.8 mg/kg bw/day. This minor change was considered to be toxicologically not relevant as all individual values remained well within the historical control ranges.

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

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

The copulatory index was lowered in male animals at 1.3 mg/kg bw/day as one male failed to mate. Pseudo-pregnancy of their female partner caused failure of mating.
Statistical significance with respect to the control was detected at the slightly lower fertility index of male and female animals (lower percentage of fertile pairing) at 1.8 mg/kg bw/day as pairing of two pairs was infertile. Therefore, the percentage of pregnant female animal was also lower than in the control at 1.8 mg/kg bw/day. Test item effect was not presumed because data met well historical control value and in the lack of related findings in the reproductive parameters or reproductive organs.
 
The copulatory and gestation indices were 100 % in the female animals in the control, 0.9, 1.3 and 1.8 mg/kg bw/day. That indicates, all female animals mated, and all pregnant female and gave live birth pups in each group.

Reproductive function: sperm measures:

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).

Reproductive performance:

no effects observed

Description (incidence and severity):

The copulatory index was lowered in male animals at 1.3 mg/kg bw/day as one male failed to mate. Pseudo-pregnancy of their female partner caused failure of mating.

Effect levels (P0)

1

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

2

Dose descriptor:

NOAEL

Effect level:

ca. 1.8 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result

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

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs were observed with low and variable incidence in the control, 0.9, 1.3 and 1.8 mg/kg bw/day groups:
- cold body temperature: 7 %, in control group;
- no milk in the stomach: 2 % in the control group, 8, 1, 1 % at 0.9, 1.3 and 1.8 mg/kg bw/day, respectively;
These signs are commonly observed in offspring of not treated dams of this strain and showed no dose dependency in this study. Therefore, these observations were considered to have no toxicological relevance.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean litter weights, mean pup weights as well as the mean litter weight gain and mean pup weight gains were similar in the control and in all test item treated groups (0.9, 1.3 and 1.8 mg/kg bw/day) on post-natal days 0, 4 and 13.
Evaluating the body weight separately by gender, the mean body weight of male and female pups was slightly lower than in the control at 1.3 mg/kg bw/day on post-natal day 4. The body weight of male and female pups was similar to their control in the low and high dose groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not specified

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

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1.8 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

other: na

Organ:

other: na

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Applicant's summary and conclusion

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 post-natal 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 post-natal 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.