5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Jun 2022 to 21 Mar 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione

EC Number:

276-763-0

EC Name:

5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione

Cas Number:

72676-55-2

Molecular formula:

C4-H2-N4-S6

IUPAC Name:

5,5'-dithiodi-1,3,4-thiadiazole-2(3H)-thione

Test material form:

solid

Test animals

Species:

rat

Strain:

other: Crl: WI (Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Time-mated female Wistar Han rats
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 178-225 g
- Fasting period before study: No
- Housing: Polycarbonate cages containing sterilized wooden fibers as bedding material equipped with water bottles. Animals were individually housed. For psychological/environmental enrichment and nesting material, animals were provided with paper and with aspen wooden sticks, except when interrupted by study procedures/activities.
- Diet: ad libitum, pellets of SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany.
- Water: ad libitum, municipal tap water
- Contamination: It is considered that there were no known contaminants in the feed, water or enrichment materials that would interfere with the objectives of the study.
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 19 °C
- Humidity: 50 to 80%
- Air changes: At least 10 air changes per hour
- Photoperiod: 12-hours light and 12-hours dark (may be interrupted for designated procedures)

IN-LIFE DATES: From: 6 June 2022 To: 24 June 2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were homogenized to visually acceptable levels, stored at room temperature and prepared within two hours prior to dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and test material. No correction was made for the purity/composition of the test material.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle chosen as it was previously used in the OECD 422 performed in the same species by oral route (gavage).
- Amount of vehicle (if gavage): 4 mL/Kg bw
- Concentration in vehicle: 25, 75 or 250 mg/mL
- Specific gravity: 0.91

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis as indicated in table 1 ''Any other information on materials and methods incl. tables''.
All samples to be analyzed were transferred (at room temperature) to the analytical laboratory at the Test Facility for same day analysis.
Concentration and homogeneity analyses were performed using a validated analytical procedure (Test Facility Study No. 20354298).

Acceptance criteria:
For concentration, mean sample concentration results within or equal to ± 10% for solutions and ± 15% for suspensions of theoretical concentration.
For homogeneity, relative standard deviation (RSD) of concentrations of = 10% for each group.

The stability of the material in peanut oil was not determined, since the available analytical method (US/Vis spectrophotometry) was not capable of indicating stability. Details of all attempts made during validation and development of the analytical method are extensively described in the analytical report (Test Facility Study No. 20354298). To limit the impact, all preparations were used within 2 hours after completion of the preparation of the formulation. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

Details on mating procedure:

- Impregnation procedure: Untreated females were mated at the Supplier and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).

Duration of treatment / exposure:

Day 6 to Day 20 post-coitum

Frequency of treatment:

Once daily

Duration of test:

Day 0 post coitum: day of successful mating
Day 21 post coitum: scheduled euthanasia

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

22 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a Dose Range Finding Study by Oral Gavage in Pregnant Wistar-Han Rats (Test Facility Study No. 20354304), and in an attempt to produce graded responses to the test material. In this study, treatment with 1000 mg/kg bw/day was generally well supported, with one animal showing erected fur on Day 20 post-coitum. This dose-level resulted in lower body weight at end of treatment (-7% compared to control) and almost absent gravid uterus adjusted body weight gain (0.78 g vs 24.54 g in control). Moreover, food consumption was decreased during the whole Treatment Period (-18% overall food consumption when compared to control). Based on the outcome of the preliminary study, the highest dose level was selected as 1000 mg/kg bw/day, as it is the limit dose for the OECD TG 414, and it did not induce death nor severe suffering.
- Fasting period before blood sampling for (rat) dam thyroid hormones: Not fasted.
- Time of day for (rat) dam blood sampling: Sampled between 07.00 and 09.00 from the jugular vein.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily 0 to 1 hours post-dose, starting on Day 6 post coitum up to and including the day prior to necropsy. All animals were also checked at least twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Regular basis throughout the study. by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 by carbon dioxide inhalation.
- All animals (including animals found dead or sacrificed before planned necropsy and females with delivery prior to necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in the appropriate fixative.
- Organ weight: the thyroid gland was weighed at necropsy for all scheduled euthanasia animals, except for females that delivered their offspring prior to necropsy. Organ weights were also not recorded for animals found dead, euthanized in poor condition or in extremis. Paired organs were weighed together.

HISTOLOGY AND MICROSCOPIC EVALUATION
- Thyroid gland of all animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin.
- The thyroid gland was examined microscopically

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes, except for animals found dead, sacrificed before planned necropsy or that started to deliver.
- Number of corpora lutea: Yes
- Number of implantations: Yes, in case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- Number and distributions of early and late resorptions: Yes
- Number and distribution of live and dead fetuses: Yes
- The sex of each fetus: Yes (based on the anogenital distance)
- Placental morphology
- Placental weights of live fetuses only of scheduled necropsy animals (after weighing,
placentae were not be retained)

Blood sampling:

- Serum: Yes, blood was sampled and processed to serum for hormone analysis.
- Volume collected: 1.0 mL
- The following parameters were measured: T3, T4, and TSH. Measurement of TSH was performed using the IMMULITE® 1000 analyser. Measurement of T3 and T4 were performed using an LC-MS system.

Fetal examinations:

- Live fetuses were euthanized by administration of sodium pentobarbital.

- External examinations: Yes: all
- Body weight: Yes: viable and non-viable fetus of dams surviving until scheduled necropsy
- Anogenital distance of all live rodent pups: Yes: all viable fetus of dam surviving until scheduled necropsy
- External sex determination: Yes: viable and non-viable fetus of dams surviving until scheduled necropsy

- Soft tissue examinations: Yes: ~50% of the fetuses (of dams surviving until scheduled necropsy)
- Skeletal examinations: Yes: ~50% of the fetuses with head (of dams surviving until scheduled necropsy)
- Head examinations: Yes: ~50% of the fetuses (of dams surviving until scheduled necropsy)
- Internal sex confirmation: Yes: 100% of the fetuses (of dams surviving until scheduled necropsy)

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons will be
conducted using two sided tests and were reported at the 1% and 5% levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Analyses were performed according to table 2 under ''Any other information on materials and methods incl. tables'' but excluded any group with less than 3 observations.

PARAMETRIC/NON-PARAMETRIC: Levene’s test was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons was conducted using Dunnett’s or Dunn’s test, respectively.

NON-PARAMETRIC: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test. The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons was conducted using Dunnett’s test.

INCIDENCE: A Fisher’s exact test was used to conduct pairwise group comparisons of interest.

Indices:

- Body weight gains: Calculated for the following intervals: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21 and 6 to 21 post-coitum.
- Gravid uterus adjusted body weight: Body weight on Day 21 post-coitum - body weight on Day 6 postcoitum - gravid uterus weight
- Overall food consumption: Calculated between each scheduled interval (individual data only) and as specified above for body weight gains. Summarization and statistical analysis intervals will reflect the same intervals as the body weight gains.
- Pregnancy rate (%): (No. of pregnant females/No. of mated females) x 100
- Organ weight relative to body weight: Calculated against body weight recorded on Day 21 post-coitum

- Live male fetuses (%): (No. of live male fetuses/No. of live fetuses) x 100
- Live female fetuses (%): (No. of live female fetuses/No. of live fetuses) x 100
- Pre-implantation loss (%): (No. of corpora lutea – No. of implantations/No. of corpora lutea) x 100
- Post-implantation loss (%): (No. of implantations – No. of live fetuses/No. of implantations) x 100
- Litter % of fetuses with abnormalities: (No. of fetuses in litter with a given finding/No. of fetuses in litter examined) x 100

Historical control data:

(1) Historical control data for pregnant Wistar Han rats (2020-2022):
T3 (ng/mL): mean (P2.5 – P97.5) = 0.424 (0.270 – 0.683) (n=347)
T4 (ng/mL): mean (P2.5 – P97.5) = 23.40 (15.07 - 45.67) (n=347)
TSH (mU/L): mean (P2.5 – P97.5) = 0.3272 (0.0838 – 0.9033) (n=392)

(2) Historical control data for pregnant Wistar Han rats (2016-2020, n = 468):
Thyroid organ weight: mean (P5-P95) = 0.0154 (0.0101 – 0.0226)
Thyroid organ/body weight: mean (P5-P95) = 0.0048 (0.0031 - 0.0069)

(3) Historical control data for pregnant Wistar Han rats (2020-2022, n=420):
Early resorptions (N): mean (±2SD) = 0.44 (0.04-0.84)
Late resorptions (N): mean (±2SD) = 0.00 (0.00-0.00)
Postimplantation loss (%): mean (±2SD) = 4.15 (0.20-0.00)
Live fetuses (N): mean (±2SD) = 11.0 (9.69 – 8.10)

(4) Historical control data for body weight of Wistar Han fetuses (period 2020-2022, n=4818 fetuses):
Males (g): mean (±2SD)= 5.295 (5.010 – 5.580)
Females (g): mean (±2SD)= 5.026 (4.809 – 5.242)
All (g): mean (±2SD)= 5.164 (4.920 – 5.408)

(5) Historical Control Data for Fetal Malformation or Variations in Wistar Han fetuses (period 2020-2022), 2316 fetuses (419 litters) skeletally examined.
Absent renal papilla: not present in historical control data
Distended urinary bladder: not present in historical control data
Convoluted ureter: mean (min-max): 3.54 (0.00-18.18); 18 fetuses (15 litters).
Dilated ureter: mean (min-max): 1.17 (0.00-5.00); 6 fetuses (5 litters).

(6) Historical Control Data for Fetal Malformation or Variations in Wistar Han fetuses (period 2020-2022), 2311 fetuses (420 litters) skeletally examined.
Wavy rib: mean (min-max): 50.14 (23.81-76.19); 428 fetuses (210 litters).
Unossified forelimb metacarpal: mean (min-max): 0.95 (0.00-9.52); 9 fetuses (4 litters).
Incomplete ossification of sternebra: mean (min-max): 3.12 (0.00-9.52); 13 fetuses (13 litters).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Erected fur was noted on 1-3 days towards the end of treatment for 1/22 and 7/19 females in the control group and at 1000 mg/kg bw/day, respectively. Moreover, hunched posture was observed in 4/19 females at 1000 mg/kg bw/day for 1-2 days towards the end of the treatment period.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test material.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female at 1000 mg/kg bw/day was sacrificed in extremis on Day 12 post-coitum, because of 16% body weight loss between Day 9 and Day 12 post-coitum. Food consumption was almost absent (down to 4 grams/day) in this period and the animal was noted with erected fur and/or hunched posture on Days 10-12 post-coitum. At necropsy, no abnormalities were found and the animal was pregnant with normal implantations for the duration of pregnancy. This poor general condition, resulting in sacrifice of the animal, was considered test material-related.

Another female at 1000 mg/kg bw/day was found dead on Day 21 post-coitum. The animal had a low food consumption from start of treatment onwards and lost 6% of its body weight over Days 6-9 post-coitum and almost 10% over Days 18-21 post-coitum. On Days 8-20 post-coitum the animal was observed with erected fur and with hunched posture on Day 20 post-coitum. At necropsy, autolysis of the whole body was noted and the animal was pregnant and had 13 dead fetuses in utero. Based on these findings, this death was considered test material-related.

A third female at 1000 mg/kg bw/day) was sacrificed on Day 20 post-coitum, because it started to deliver its offspring that day, which is considered as abortion. The animal had almost no body weight gain over Days 15-18 post-coitum and lower food consumption (7 grams/day) over Days 15-18 post coitum. Furthermore, the animal was noted with erected fur, hunched posture, red discharge around muzzle and was scored hypersensitive on Day 19 post-coitum. At necropsy, enlargement and dark red foci of the adrenal glands were noted and a small thymus was observed. Moreover, minimal diffuse follicular cell hypertrophy of thyroid gland was found. This animal had one late resorption and 9 live fetuses in utero and 4 live fetuses ex utero. As this animal was in bad condition before the animal started to deliver her litter, this abortion, resulting in sacrifice of the animal, was considered test material-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights, body weight gain and gravid uterus adjusted body weight gain remained in the same range as control over the treatment period up to 300 mg/kg bw/day.
At 1000 mg/kg bw/day, lower body weight gain was noted for pregnant animals from start of treatment onwards (reaching significance from Day 15 post-coitum onwards, with the exception over Days 12-15 post-coitum. This resulted in a 17% lower (statically significant) mean body weight on Day 21 post-coitum. Furthermore, the mean gravid uterus weight was 19% lower (statistically significant) and there was a near absent gravid uterus adjusted body weight gain (0.37 g vs. 32.58 g in control, 7/17 females had gravid uterus adjusted body weight loss).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 100 mg/kg bw/day, food consumption was 10% lower than control over Days 18-21 post-coitum (not statistically significant).
At 300 mg/kg bw/day, slightly lower food consumption (3-5%, not statistically significant) was noted over Days 9-18 post coitum, followed by statistically significant 10% lower food consumption over Days 18-21 post-coitum. Overall food intake during the treatment period was 5% lower than control (not statistically significant).
At 1000 mg/kg bw/day, mean food consumption was lower throughout the entire treatment period (statistically significant for every interval). The largest difference was observed during the last measurement interval (i.e., Days 18-21 post-coitum) where food intake was 56% lower than control. Overall food intake during the treatment period was 25% lower (statistically significant) than control.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormone levels for all treatment groups were summarized in table 3 under ''Any other information on results incl. tables''
Up to 300 mg/kg bw/day, serum levels of T3 were considered to be unaffected by treatment with the test material.
Serum levels of T3 were lower at 1000 mg/kg bw/day (0.46x of control, with 4/20 females with level below the Limit of Quantification of 0.1 ng/mL). Given the magnitude of the effect, a test material-related effect could not be excluded. The mean value was below the historical control data.
Serum levels of T4 were lower at 100 and 300 mg/kg bw/day (0.71 and 0.52x of control, respectively) and were undetectable at 1000 mg/kg bw/day (below the Limit of Quantification of 5 ng/mL). The lower T4 level is considered test material related in all groups. Although the mean value at 100 mg/kg bw/day remained within historical control data, the mean value at 300 mg/kg bw/day was below the historical control data.
Serum levels of TSH were higher at 100, 300 and 1000 mg/kg bw/day (1.81, 2.94 and 19.06x of control, respectively), which was considered test material related. The mean value at 100 mg/kg bw/day was within the historical control data, but the mean values at 300 and 1000 mg/kg bw/day were above the historical control data.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Dose-dependent higher thyroid gland weights (absolute and relative to body weight) were noted. At 1000 mg/kg bw/day, significance was reached and the mean value for thyroid gland weight relative to body weight was above the higher limit of the historical control data.
Please see table 4 under ''Any other information on results incl. tables''.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test material-related gross observations in the thyroid gland of rats treated at up to 300 mg/kg bw/day.
There were gross observations regarded test material-related in the thyroid glands of rats at 1000 mg/kg bw/day. These included bilateral enlargement in 7/22 females and/or bilateral dark red discoloration in 3/22 females.
The unilateral enlargement of the thyroid gland in a single female at 300 mg/kg bw/day was without microscopic correlate and regarded within background.
A small thymus was noted in 7/22 females at 1000 mg/kg bw/day.
Other findings that were noted among treated animals were considered to be unrelated to treatment with the test material, as they remained within the range of biological variation for rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings in the thyroid gland after treatment with the test material were noted at 1000 mg/kg bw/day group females and are summarized in table 5 under ''Any other information on results incl. tables''.
Diffuse follicular cell hypertrophy at an increased incidence and severity was noted at 1000 mg/kg bw/day. This was seen in 18/21 animals at minimal to moderate degree.
The minimal severity of diffuse follicular cell hypertrophy in the concurrent controls and the few other microscopic findings recorded in the thyroid gland of one female at 300 mg/kg bw/day were regarded within background. There was no test material related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

One additional female at 1000 mg/kg bw/day was sacrificed on Day 20 post-coitum due to abortion.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, higher mean postimplantation loss at this dose level (16.7% vs. 5.13% in control) at 1000 mg/kg bw/day. Mean values for post-implantation loss were above the historical control range.
At 300 mg/kg bw/day, there was a slightly higher mean postimplantation loss (7.61% vs. 5.13% in control). Although the mean post implantation loss was within the historical control range.
See also tables 6 under ''Any other information on results incl. tables''

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One high dose females was observed with late resorptions only (13 implantations)

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, an increased mean number of early resorptions (1.0 vs. 0.6 in control) and late resorptions (0.9 vs. 0.0 in control). Mean values for early and late resorptions were above the historical control range.
At 300 mg/kg bw/day, the mean number of early resorptions was increased (0.9 vs. in 0.6 in control), resulting in slightly higher mean postimplantation loss (7.61% vs. 5.13% in control). The number of early resorptions was above the historical control range.
See also table 6 under ''Any other information on results incl. tables''

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

One fetus from a high dose female was found dead.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

All females, except two at 1000 mg/kg bw/day were pregnant.

Details on maternal toxic effects:

Excluding non-pregnant females and females that did not survive until scheduled necropsy, there were 22, 22, 22 and 17 females available for ovarian and uterine examination at scheduled necropsy in the control, 100, 300 and 1000 mg/kg bw/day groups, respectively.
The numbers of corpora lutea, implantations sites and pre-implantation loss in control and test material-treated groups were within the same range and in the range of normal biological variation.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

endocrine findings

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

pre and post implantation loss

Maternal abnormalities

Abnormalities:

not specified

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no test material-related effects on fetal body weights noted at up to 300 mg/kg bw/day.
At 1000 mg/kg bw/day, mean fetal body weights were 14% lower (statistically significant) than control. Mean values for body weight were below the available historical control data range.
See also table 7 under ''Any other information on results incl. tables''

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered to be unaffected by treatment with the test material.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

There were no test material-related effects on litter size up to 300 mg/kg bw/day. Mean litter sizes were 11.0, 10.9 and 10.9 fetuses for control, 100 and 300 and mg/kg bw/day treated animals, respectively.
At 1000 mg/kg bw/day, mean litter size was lower (9.5 vs. 11.0 in control, not statistically significant), and the mean value was below the lower limit of the historical control data.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean fetal anogenital distance was considered to be unaffected by treatment with the test material.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related external malformations and variations were recorded.
In this study one external malformation was observed in a mid-dose fetus (300 mg/kg bw/day), which presented with exencephaly. This single occurrence was considered a chance finding.
See also tables 6 and 8 under ''Any other information on results incl. tables''

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No test material-related skeletal malformations were observed.
In this study two fetuses (one at 300 mg/kg bw/day and another at 1000 mg/kg bw/day) presented with skeletal malformations. The fetus from the mid-dose group (300 mg/kg bw/day) had skeletal malformations that corresponded to its external finding of exencephaly. Due to the single occurrences of these skeletal malformations in context to a single external finding, a relationship to treatment with the test material was excluded. Additionally, the high-dose fetus (1000 mg/kg bw/day) had an absent lumbar vertebra the single occurrence of which, was ruled a chance finding.
Skeletal variations were observed in the forelimb, pelvic girdle, (supernumerary) ribs, skull, sternebra and vertebra. Among the skeletal variations there were significantly fewer incidences of wavy ribs observed in the low- and high-dose groups, when compared to the control. Furthermore, observations in the mid-dose group (300 mg/kg bw/day) were also fewer in number when compared to the control. While the difference was not significant, it was beneath the range of historical control data (6). Hence, the decrease in observations of wavy ribs at all dose levels was considered related to the treatment with the test material.
In addition, the low-dose group was observed to have higher incidences of incomplete/unossified bones (e.g., unossified metacarpals and incompletely ossified sternebra). Mean fetal incidences exceeded the range of historical control data (6). However, there was no significant difference from the control and due to an absence of a dose response, a relationship to treatment with the test material was ruled out. In all other cases, variations were observed either infrequently, in incidences comparable to the control group and/or in the absence of a dose-related incidence trend. Therefore, considered not to be related to treatment with the test material.
See also tables 6 and 10 under ''Any other information on results incl. tables''

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

No test material-related visceral malformations were observed in this study.
In this study, two fetuses at 300 mg/kg bw/day presented with visceral malformations. The majority of visceral malformations occurred in the control group fetus, which had misshapen heart, ventricular septal defect, transposition of great vessels, malpositioned vena cava and absent lung lobe. As these malformations were noted in the control group, they were spontaneous in origin. The mid-dose group fetus (300 mg/kg bw/day) had a retroesophageal, right subclavian artery. This single occurrence was considered a chance finding.
Visceral variations observed in this study affected the innominate artery, kidney, liver, ureter and urinary bladder. The most prominent findings afflicted the urinary system with incidences of absent renal papilla, distended bladders and dilatated ureters that were all significantly higher in the high-dose group (1000 mg/kg bw/day) when compared to the control.
Furthermore, the occurrence of convoluted ureters was only present in test material-treated fetuses, but remained within the historical control range for all groups. However a relationship with test material could not be excluded for the findings regarding the urinary system.
All other visceral variations were observed in instances comparable to the control or in the absence of a dose related incidence trend. Therefore, these were considered not to be related to treatment with the test material.
Two incidental findings of discoloration (liver) were observed in the low- and mid-dose group. However, these were considered chance findings.
See also tables 6 and 9 under ''Any other information on results incl. tables''

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The placental weights were considered to be unaffected by treatment with the test material up at to 300 mg/kg bw/day.
At 1000 mg/kg bw/day, mean placental weights were 14, 11 and 13% lower than control , for males, females and overall, respectively.

Details on embryotoxic / teratogenic effects:

The numbers of fetuses (litters) from the control, low-, mid-, and high-dose groups respectively submitted to the different examinations, were as follows:

External examination: 243 (22), 239 (22), 240 (22) and 162 (16)
Visceral examination: 120 (22), 120 (22), 120 (22) and 81 (16)
Skeletal examination: 123 (22), 119 (22), 120 (22) and 81 (16)

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

Develpmental

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

other: Reduced placental weight

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: urinary

Description (incidence and severity):

Se developmental results section

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

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

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

 

DOSE FORMULATION ANALYSIS:

Accuracy: The concentrations analysed in the formulations of Groups 2, 3 and 4 in both Week 1 and Week 2 were in agreement with target concentrations (i.e., mean sample concentration results were within or equal to 85-115% of target concentration). A small response was observed in both the Group 1 formulation prepared for use in Week 1 and Week 2. The maximum contribution of this small response to the Group 2 samples was 0.23% in Week 1 and 0.21% in Week 2. As the maximum contribution was <1% to the Group 2 samples, it was concluded that this small response had no impact on the outcome of the study.

Homogeneity: The formulations of Groups 2 and 4 in both Week 1 and Week 2 were homogeneous (i.e., coefficient of variation ≤ 10%).

 

Table 3. Summary of Thyroid hormone levels (fold change compared to control)

Dose level (mg/kg bw/day)	0	100	300	1000	Historical control data pregnant Wistar Han rats (2020-2022)
T3 (ng/mL)	0.453	0.433 (0.98x)	0.397 (0.88x)	0.210** (0.46x)	0.424 (0.270 – 0.683)
T4 (ng/mL)	25.22	17.98** (0.71x)	13.21** (0.52x)	< 5.00	23.40 (15.07 - 45.67)
TSH (mU/L)	0.3428	0.6220* (1.81x)	1.0085** (2.94x)	6.5325**  (19.06x)	0.3272 (0.0838 – 0.9033)

* p ≤ 0.05; ** p ≤ 0.01

 

Table 4. Mean Percent Thyroid Gland Weight Differences from Control Group

Dose level (mg/kg bw/day)	0	100	300	1000	Historical control data pregnant Wistar Han rats (2016-2020)
THYROID GLANDS
Absolute	0.0144	0.0147 (2%)	0.0156 (9%)	0.0210 (46%)**	0.0154 (0.0101- 0.0226)
Relative to body weight	0.0043	0.0045 (5%)	0.0048 (11%)	0.0076 (77%)**	0.0048 (0.0031 - 0.0069)

** = P≤0.01

 

Table 5. Summary Test Material-Related Microscopic Findings Thyroid Gland (excluding female that was found dead)

Dose level (mg/kg bw/day)	0	100	300	1000
THYROID GLANDS ^(a)	22	22	22	21
Hypertrophy follicular cell, diffuse
Minimal	5	-	-	2
Mild	-	-	-	8
Moderate	-	-	-	8

^a = Number of tissues examined from each group.

 

Table 6. Overall summary of reproductive parameters

Dose level (mg/kg bw/day)	0	100	300	1000
Pregnant/total dams	22/22	22/22	22/22	20/22
Dams with abortion, early deliveries, stillbirths, resorptions only and/or dead fetuses	0	0	0	2
Dams with live fetuses	22	22	22	18
Corpora lutea (mean number)	12.5	12.5	12.4	12.4
Implantations (mean number)	16.6	11.3	11.8	11.5
Resorptions (mean number) - Early - Late - Total (early + late)	0.6 0.0 0.6	0.5 0.0 0.5	0.9 0.0 0.9	1.0 0.9 1.9
Pre implantation loss (%)	7.22	9.91	5.26	6.35
Post implantation loss (%)	5.13	4.70	7.61	16.71
Dead fetuses (mean number)	0.0	0.0	0.0	0.1
Mean body weight on day 21 (g)	333.7	327.0	327.6	277.2**
Mean body weight gain day 6-21 (g)	110.2	106.0	104.6	63.5**
Gravid uterine weight (g)	77.60	74.28	75.84	63.16*
Mean adjusted body weight gain (6-abw)	32.58	31.72	28.80	0.37**
Live offspring (mean number)	11.0	10.9	10.9	9.5
Sex ratio (male%)	50.52	48.52	41.83	40.33
Mean anogenital distance males (mm)	2.94	2.91	2.92	2.88
Mean anogenital distance females (mm)	1.52	1.56	1.56	1.59
Mean normalized anogenital distance males	1.683	1.695	1.665	1.744
Mean normalized anogenital distance females	0.884	0.920	0.904	0.977
Malformations number (and % of fetuses) number of litters - External - Visceral - Skeletal	0 (0.0%) 0 1 (0.76%) 1 0 (0.0%) 0	0 (0.0%) 0 0 (0.0%) 0 0 (0.0%) 0	1 (0.38%) 1 1 (1.14%) 1 1 (0.76%) 1	0 (0.0%) 0 0 (0.0%) 0 1 (1.56%) 1
Variations number and % of fetuses number of litters - External - Visceral - Skeletal	0 (0.0%) 0 5 (5.08%) 4 86 (69.42%) 22	0 (0.0%) 0 15 (11.34%) 8 94 (74.42%) 21	0 (0.0%) 0 17 (15.54%) 11 93 (78.19%) 22	0 (0.0%) 0 16 (20.10%) 10 51 (60.91%) 15

*  = p ≤ 0.05
** = p ≤ 0.01

 

Table 7. Summary of mean Fetal Body Weights (Percentual Difference Compared to Control)

Dose level (mg/kg bw/day)	0	100	300	1000	Historical control data pregnant Wistar Han rats (2020-2022)
Mean Fetal Weight Males (gram)	5.331	5.167 (-3%)	5.402 (1.3%)	4.588 (-14%)**	5.295 (5.010- 5.580)
Mean Fetal Weight Females (gram)	5.081	4.940 (-3%)	5.115 (1%)	4.387 (-14%))**	5.026 (4.809- 5.242)
Mean Fetal Weights Males and Females Combined (gram)	5.200	5.051 (-3%)	5.234 (1%)	4.482  (-14%)**	5.164 (4.920 - 5.408)

**: P≤0.01

 

Table 8. Summary of External Malformations - Individual Descriptions

Dose Level (mg/kg bw/day)	No. of fetuses	Malformation(s)#
300	one fetus	Head/neck, Exencephaly

 

Table 9. Summary of Visceral Malformations - Individual Descriptions

Dose Level (mg/kg bw/day)	No. of fetuses	Malformation(s)#
0	one fetus	Great Vessels, Transposition of Great Vessels
		Heart, Misshapen
		Heart, Muscular Ventricular Septal Defect
		Lung Lobe, Accessory, Absent
		Posterior (caudal) vena cava, Malpositioned
	1 from a differnt litter	Subclavian artery, Right, Retroesophageal

 

Table 10. Summary of Skeletal Malformations - Individual Descriptions

Dose Level (mg/kg bw/day)	No. of fetuses	Malformation(s)
300	one fetus.  The same fetus had head/neck, exencephaly as external malformation	Frontal, Misshapen
		Interparietal, Misshapen
		Parietal, Misshapen
		Supraoccipital, Split
1000	one fetus	Lumbar centrum, Absent

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study in time mated female Wistar Han rats the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Bis DMTD were established:

Maternal NOAEL: 300 mg/kg bw/day (based on mortality, lower body weight, lower body weight gain, lower gravid uterus adjusted body weight gain, lower food intake, pathomorphological alterations of the thyroid and thyroid hormones levels, and higher postimplantation loss at 1000 mg/kg bw/day).
Developmental NOAEL: 300 mg/kg bw/day (based on lower litter size and reduced fetal body and placental weights at 1000 mg/kg bw/day).

A reduction of the serum T4 level and increased serum TSH level were observed at 300 mg/kg bw/day which was considered to be test material-related. However, possible adversity of these effects could not be assessed within this type of study and was therefore not taken into account when determining the maternal NOAEL.
Developmental effects in this study were observed at a maternal toxic dose. It could not be excluded that these effects were secondary to the maternal condition and not a direct effect of the test material.

Executive summary:

The objectives of this study were to determine the potential of 5,5'-dithiobis-(1,3,4-thiadiazole-2-thiol) (Bis DMTD) to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.
The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg/day, based on the results of the Dose Range Finder.
Chemical analyses of formulations were conducted twice during the study and confirmed that formulations of test material in peanut oil were prepared accurately and homogenously.
The following parameters and end points were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, thyroid hormone levels (T3, T4, TSH), organ weights (thyroid gland), macroscopic examination, microscopic examination (thyroid gland) and uterine contents (including corpora lutea, implantation sites, pre- and post-implantation loss and number of live and dead fetuses).
In addition, the following parameters were determined for the F1-generation: fetal body weights, sex ratio, anogenital distance, placental weights, external, visceral and skeletal malformations and developmental variations.
One female at 1000 mg/kg/day was sacrificed in extremis on Day 12 post-coitum based body weight loss, reduced food consumption and clinical signs. Furthermore, one female at 1000 mg/kg/day was found dead on Day 21 post-coitum. The poor general condition of these animals, resulting in sacrifice of the animals, was considered test material-related. One additional female at 1000 mg/kg/day was sacrificed on Day 20 post-coitum due to abortion. As this female had clinical signs, almost absent body weight gain and food consumption on the days prior delivery, this abortion was considered test material-related.
No maternal toxicity was observed at 100 mg/kg/day. The significantly lower T4 (0.71x of control) and higher TSH (1.81x of control) levels at this dose-level were considered non adverse as mean values remained within historical control range.
At 300 mg/kg/day, slightly lower food consumption was noted overall during the treatment period (-5% vs. control, not significant). Moreover, postimplantation loss was slightly higher (7.61% vs. 5.13% in control). Both effects were considered non-adverse. Test-material related significantly lower T4 serum levels (0.52x of control) and higher TSH levels (2.94x of control) were noted at 300 mg/kg/day, with mean values outside the normal range of variation. Possible adversity of these effects at this dose could not be assessed within this type of study.
At 1000 mg/kg/day, significantly lower body weight gain was noted from Day 15 post-coitum onwards, which resulted in significantly lower body weight at Day 21 post-coitum (-17% vs. control). Furthermore, there was a near absent gravid uterus adjusted body weight gain (0.37 g vs. 32.58 g in control, 7/17 females had gravid uterus adjusted body weight loss).. Correlating progressive significantly lower food consumption (-25% vs. control, overall) was observed and increased postimplantation loss (16.7% vs. 5.13% in control) was seen for these females. These effects were considered adverse.
At 1000 mg/kg/day, an increased incidence and severity of diffuse follicular cell hypertrophy was noted. This was accompanied by significantly higher thyroid glands weights (absolute: +46% vs. control; relative: +77% vs. control) and the macroscopic enlargement of the thyroid glands in this dose group. The follicular cell hypertrophy correlated to the alterations in thyroid hormones, including significantly lower T3 (0.46x of control, with 4/20 females with level below the Limit of Quantification of 0.1 ng/mL) and T4 (below the Limit of Quantification of 5 ng/mL) and higher TSH (19.06x of control) in females at 1000 mg/kg/day. The combination of all these observations was considered adverse at this dose level.
No test material-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e., clinical appearance, uterine contents including corpora lutea, implantation sites and pre-implantation loss).
No developmental toxicity was observed in the 100 and 300 mg/kg/day groups.
At 1000 mg/kg/day, adverse reduced fetal body (-14% vs. control, significant) and placental weights 13% vs control, significant) were noted. Moreover, litter size was adversely lower at this dose level (9.5 vs. 11.0 in control, not significant), due to increased postimplantation loss.
The increased incidence in visceral variations regarding the urinary tract (i.e., absent renal papilla, distended bladders, dilatated ureters, convoluted ureters) at both 300 and 1000 mg/kg/day and the lower incidence of wavy ribs in all treatment groups were all considered non-adverse.
No test material-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e,. sex ratio, anogenital distance, external, visceral and skeletal malformations, and incidental findings).
In conclusion, based on the results of this prenatal developmental toxicity study in time mated female Wistar Han rats the following maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Bis DMTD were established:
Maternal NOAEL: 300 mg/kg/day (based on mortality, lower body weight, lower body weight gain, lower gravid uterus adjusted body weight gain, lower food intake, pathomorphological alterations of the thyroid and thyroid hormones levels, and higher postimplantation loss at 1000 mg/kg/day).
Developmental NOAEL: 300 mg/kg/day (based on lower litter size and reduced fetal body and placental weights at 1000 mg/kg/day).
A reduction of the serum T4 level and increased serum TSH level were observed at 300 mg/kg/day which was considered to be test material-related. However, possible adversity of these effects could not be assessed within this type of study and was therefore not taken into account when determining the maternal NOAEL.
Developmental effects in this study were observed at a maternal toxic dose. It could not be excluded that these effects were secondary to the maternal condition and not a direct effect of the test material.