1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

aug-nov 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD and EU guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421, Reproduction/Developmental Toxicity Screening Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

EC Number:

700-464-0

Cas Number:

99591-74-9

Molecular formula:

C2H4O6S2

IUPAC Name:

1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

Constituent 2

Reference substance name:

MMDS

IUPAC Name:

MMDS

Details on test material:

- Name of test material (as cited in study report): MMDS
- Substance type: powder
- Physical state: solid

Test animals

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 11 weeks
- Housing: Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages; Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages; Post-mating: Males were housed in their home cage (Macrolon plastic cages) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages; Lactation: Pups were kept with the dam until termination in Macrolon plastic cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 19.7 – 21.5°C)
- Humidity (%): a relative humidity of 40 - 70% (actual range: 44 - 77%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 October 2011 To: 25 November 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

specific gravity 0.92 (Sigma-Aldrich Steinheim Germany)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose volume: 2 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
The test substance and the formulation were handled as much as possible under nitrogen using a glove box. Formulations of Groups 2-4 (w/w) were prepared within 15 minutes prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and on information provided by the sponsor.

Details on mating procedure:

Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Preparation of formulations was considered acceptable if the mean accuracy was in the range
85-115% of the target concentration and if the coefficient of variation was ≤ 10%.
In the Group 1 formulations, no test substance was detected. The concentrations analysed in the formulations of Group 2 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The concentrations analysed in the formulations of Group 3 were at 77% and 82% of the target concentration. This is slightly outside the criterion of 85 - 115% but still considered acceptable for this difficult to analyse substance.

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

For details on the analytical method see section 8.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 43 to 53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Females 44, 48 (Group 1) and 70 (Group 3) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 40, 200 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 10-day dose range finding study (NOTOX Project 497701)

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily 2 hours after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Yes, weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of FOB

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters checked: according to guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters checked: according to guideline

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Dose groups that were examined: 5 animals/sex/group

Oestrous cyclicity (parental animals):

no data

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, seminal vesicles including coagulating glands.
From selected 5 males of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis.

Litter observations:

Each litter was examined to determine mortality/viability, clinical signs, body weights and sex.
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):

Histological examination was performed on reproductive organs.
Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:

Mating index (%) Number of females mated/ Number of females paired x 100

Fertility index (%) Number of pregnant females/ Number of females paired x 100

Conception index (%) Number of pregnant females/ Number of females mated x 100

Gestation index (%) Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/ Number of live pups at First Litter Check x 100
Viability index: Number of live pups on Day 4 post partum/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

3 high dose females died/were killed in extermis

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high dose males during the complete treatment period

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

high dose males during the complete treatment period

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

treatment-related microscopic findings in stomach, duodenum, prostate gland and mesenteric lymph node

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): one high dose female was found dead on day 15, two high dose females were killed in extremis on days 33 and 39, respectively. Clinical signs were restricted to the high dose group, and consisted of hunched posture, rales and piloerection during days 1-4 of the treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS): In high dose males deceased absolute and relative thymus weights and increased relative weights of the brain, liver, thyroids, kidneys, adrenals and testes were observed. In high dose females, increased relative liver, kidneys and adrenals weights were noted.

GROSS PATHOLOGY (PARENTAL ANIMALS): Stomach abnormalities were noted for two males and two females treated at 200 mg/kg, and consisted of reddish foci in the glandular mucosa, crateriform retraction in the forestomach and/or thickened limited ridge.
Red discolouration of the mesenteric lymph nodes were seen in four animals treated at 10 mg/kg, in eleven animals treated at 40 mg/kg and in sixteen animals treated at 200 mg/kg.

HISTOPATHOLOGY (PARENTAL ANIMALS): Stomach:
- An increased incidence and/or severity of lymphogranulocytic inflammation of the submucosa of the glandular stomach was recorded in Group 4 in 6/6 males (minimal-moderate) and 4/7 females (minimal-slight). This was also recorded in Group 2 in 2/5 males (minimal, slight) and 1/5 females (minimal) and in Group 3 in 2/5 males and 1/5 females (minimal).
- An ulcer in the forestomach was recorded in 1/6 males (moderate) accompanied by slight necrosis of the muscular layer, and in 1/7 females (marked) of Group 4.
- Hyperplasia of the squamous epithelium of the forestomach was recorded in 1/6 males (slight) and 2/7 females (minimal, moderate) of Group 4, in one female accompanied by a moderate lymphocytic inflammation of the forestomach.
- Erosion of the glandular mucosa was recorded in 2/7 females (slight) of Group 4.
Duodenum: Minimal fibrosis in the villi was recorded in 2/7 females of Group 4.
Prostate gland: An increased incidence and/or severity of lymphocytic inflammation was recorded in 1/5 males of Group 2 (moderate), 1/5 males of Group 3 (minimal) and 3/6 males of Group 4 (minimal-moderate).
Mesenteric lymph nodes: An increased incidence and severity of sinus erythrocytosis with erythrophagocytosis and histiocytosis was recorded in Group 2 and above: In Group 2 in 2/5 males (minimal) and 3/5 females (minimal-slight), in Group 3 in 7/7 males and 4/5 females (slight) and in Group 4 in 10/10 males (slight-moderate) and 7/7 females (minimal-slight). This was also seen in 1/5 females of Group 1 (minimal).

Effect levels (P0)

1

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: dose-related increase in the incidence of red discolouration of the mesenteric lymphnodes and dose-related increased incidence and/or severity of sinus erythrocytosis with erythrophagocytosis and histiocytosis

2

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1a

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity was observed up to the highest dose level tested (200 mg/kg).

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with MMDS by oral gavage in male and female Wistar Han rats at dose levels of 10, 40 and 200 mg/kg body weight/day revealed parental toxicity at all dose levels (dose-releated increase in the incidence of red discolouration of the mesenteric lymphnodes and dose-related increased incidence and/or severity of sinus erythrocytosis with erythrophagocytosis and histocytosis), and therefore no NOAEL for parental toxicity could be derived. The LOAEL for parental toxicity is 10 mg/kg bw/day. No reproduction and developmental toxicity was observed for treatment up to 200 mg/kg body weight/day. A reproduction and developmental NOAEL of at least 200 mg/kg/day was derived.

Executive summary:

MMDS was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 10, 40 and 200 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 43 to 53 days).

Parental toxicity was observed at all dose levels (10, 40 and 200 mg/kg) in a dose related manner, and consisted of dose-releated increase in the incidence of red discolouration of the mesenteric lymphnodes and dose-related increased incidence and/or severity of sinus erythrocytosis with erythrophagocytosis and histiocytosis. Based on these effects at all dose levels, a NOAEL for MMDS could not be established, and the LOAEL for parental toxicity is 10 mg/kg bw/day.

No reproduction and developmental toxicity was observed for treatment up to 200 mg/kg body weight/day. A reproduction and developmental NOAEL of ≥ 200 mg/kg/day was derived.