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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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:

other: 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)

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.

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

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.

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.

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.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Frequency of treatment:

parental animals: 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. Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.

Duration of test:

Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.

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

Maternal 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation days 5-7 (females which delivered); post-coitum day 27 (females which failed to deliver); males following completion of teh mating period (at least 28 days of dose administration).
- Organs examined: according to guideline

OTHER: FOB

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes
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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
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

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

LOAEL

Remarks:

parental

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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, up to the highest dose level tested (200 mg/kg).

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

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, up to the highest dose level tested (200 mg/kg). Based on these observations, the NOAEL for developmental toxicity of MMDS is at least 200 mg/kg bw/day.

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, duringpost-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 ofdose-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.