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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In an OECD 422 study, the NOEL for reproductive toxicity to rats of 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione is 300 mg/kg bw/day, based upon all reproductive and developmental toxicity endpoints evaluated. Adverse effects on reproduction and development noted at 1,000 mg/kg bw/day were considered to have been attributed to secondary parental toxicity.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

yes

Remarks:

Deviations are considered not to have affected the integrity or purpose of the study.

Principles of method if other than guideline:

Deviations are noted but do not affect the integrity or purpose of the study.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source:
The animals were acclimatized for eight days. A total of one hundred and twenty animals (sixty males and sixty females) were accepted into the study. At the start of treatment the males weighed 302 to 351g, the females weighed 184 to 231g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation, parturition and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Pelleted Diet) was used. Tap drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation.

The animals were housed in a single air-conditioned room. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored. The temperature remained within 22 ± 3 °C during the study. Transient deviations from the target range for relative humidity of 50 ± 20% were considered not to have affected the purpose or integrity of the study.

The animals were uniquely identified within the study by an ear punching system and a tail mark.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

The test item was prepared at the appropriate concentrations in Arachis oil BP. The homogeneity of the test item formulations were confirmed by analysis. Formulations were prepared on a daily basis; all animals were dosed within 2.5 hours after dose preparation. The formulations were considered to be stable for the period between preparation and dosing.

Test item formulation were analyzed for concentration of 5,5’-Dithiodi-1,3,4-thiadiazole-2(3H)-thione . The results indicate that the prepared formulations were within 80-107% of the nominal concentration.

Details on mating procedure:

Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined spectrophtometrically.
The detection system was found to have acceptable linearity. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven suitable for use.

Duration of treatment / exposure:

41-47 days. Males were dosed daily throughout the study until the day prior to scheduled necropsy. Females were dosed for a two week pre-pairing period, throughout pairing and gestation and early lactation (Days 1-4). Females in parturition were not dosed on that day.

Frequency of treatment:

Once daily by gavage.

Details on study schedule:

Each pregnant female was observed at least three times a day (nominally early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at early morning (approximately 0830) and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition (see Deviations from Study Plan)
iv. Date and time of observed completion of parturition (see Deviations from Study Plan)

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum (see deviations from Study Plan)
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from these data)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 males + 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

Five dose groups (control, low, intermediate I, intermediate II and high) each comprising 24 animals (12 male and 12 female) were used. Dose levels were chosen based on available toxicity data including a fourteen day range-finding toxicity study. In this range-finding study, a dosage of 1000 mg/kg bw/day was well tolerated but increases in liver and kidneys weights were apparent for both sexes and a low level of brown/yellow pigment disposition was observed in the kidneys for females. Dosages of 0 (Control), 30, 100, 300 and 1000 mg/kg bw/day were chosen in collaboration with the Study Monitor for the Sponsors.

i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals in an open arena were observed for signs of functional/behavioral changes.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages with suitable nesting material. Dosing continued through pairing and subsequent female gestation and lactation phase (females in parturition were not dosed).
v. During Week 6, five selected males per dose group were evaluated for functional/sensory responses to various stimuli, grip strength and motor activity assessments.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were recorded during this period.
vii. At Day 4 post partum or Day 25 post coitum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli, grip strength and motor activity assessments.
viii. Blood samples were taken from the same five males from each dose group for hematological and blood chemical assessments on Day 42.
ix. On Day 43, six males from each dose group were killed and examined macroscopically . The remaining six males from each dose group were weighed.
x. On Day 44, the remaining six males from each dose group were killed and examined macroscopically.
xi. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum or Day 25 post coitum for non-pregnant females. At Day 5 post partum, all females and their surviving offspring were killed and examined macroscopically. Any female which did not produce a pregnancy or litter was also killed and examined macroscopically on or after Day 25 post coitum.

Positive control:

not required

Parental animals: Observations and examinations:

See Repeated Dose toxicity, 7.5.1., Key Study for general evaluation of parental animals related to the repeat dose portion of the study.

Oestrous cyclicity (parental animals):

not observed

Sperm parameters (parental animals):

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: no

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

Necropsy
Adult animals were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination. Males were killed on Day 43 or Day 44, females that littered were killed on Day 5 post partum, females that failed to achieve pregnancy or produce a litter were killed on Day 25 post coitum, and one female was killed in extremis during parturition. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced, as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from the five selected males and five selected females from each dose group. Tissues shown with* were weighed from all remaining animals:

Adrenals
Prostate*
Brain
Seminal vesicles*
Epididymides*
Spleen
Heart
Testes*
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries*
Uterus (weighed with Cervix)*
Pituitary (post fixation)*

Histopathology
Samples of the following tissues were removed from the five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown with* were preserved from all remaining animals:

Adrenals
Muscle (skeletal)
Aorta (thoracic)
Ovaries*
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary*
Brain (including cerebrum, cerebellum and pons)
Prostate*
Caecum
Rectum
Coagulating gland*
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides*•
Skin (hind limb)
Esophagus
Eyes+
Spinal cord (cervical, mid-thoracic and lumbar)
Gross lesions
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes*•
Liver
Thymus
Lungs (with bronchi) #
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix*
Mammary gland*
Vagina*

+ = eyes fixed in Davidson’s fluid
• = preserved in Modified Davidsons fluid
# = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

Tissues were dispatched to the Test Site (Propath UK Ltd) for processing. The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown with* from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.
Following the initial results of histopathology for high dosage animals, histopathological examination was extended to five animals of either sex (except where stated) for the low and both intermediate dosage groups for the following tissues: adrenal glands (males only), lymph nodes, pituitary (all animals), salivary gland, spleen (females only), thyroid glands, kidney, liver and thymus.
Microscopic examination was conducted by the Study Pathologist and a peer review being conducted by Peter Millar Associates Ltd.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Additional data are available upon request.

Reproductive indices:

Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval

Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices

For each group the following were calculated:

Mating Index (%) = number of animals mated/number of animals paired x 100

Pregnancy Index (%) = number of pregnant females/number of animals mated x 100

The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length

Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index

The following was calculated for each group:

Parturition Index (%) = number of females delivering live offspring/number of pregnant females x 100

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)

Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss (%) = Number of corpora lutea - number of implantation sites/number of corpora lutea x100

Post–implantation loss (%) = Number of implantation sites - total number of offspring born/ number of implantation sites x100

ii. Live Birth and Viability Indices

The following indices were calculated for each litter as follows:

Live Birth Index (%) = Number of offspring alive on Day 1/number of offspring born x 100

Viability Index (%) = Number of offspring alive on Day 4/Number of offspring alive on Day 1 x 100

iii. Sex Ratio (% males)

Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

Number of male offspring/Total number of offspring x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Mortality:

mortality observed, treatment-related

Description (incidence):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Repeated Dose toxicity, 7.5.1. Key Study(2016) for general evaluation of parental animals.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day there was not effect on mating but the pregnancy rate was poor.
At 1000 mg/kg bw/day, there was no effect on mating (all 12 females mated), but the subsequent pregnancy rate was poor with three females failing to achieve pregnancy. Three of the nine pregnant females were not observed to give birth to a litter and one pregnant female was killed around the time of parturition. Two of the remaining females showed total litter loss post partum and only three females successfully reared their young to Day 5 of lactation.

There was no effect of treatment on mating and fertility at 30, 100 or 300 mg/kg bw/day.

At 1000 mg/kg bw/day, there was a clear increase in the length of gestation for the five females observed to give birth to a litter, with a mean gestation length of 24.9 days for the high dose compared to 22.5 days for the control group.

There was no effect of treatment on gestation length at 30, 100 or 300 mg/kg bw/day.

At 1000 mg/kg bw/day, assessment of any effect of treatment on corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age was frustrated by the small group size, although a clear increase in post implantation loss was apparent and supported by the incidence of post partum total litter loss observed at this dosage.

There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30, 100 or 300 mg/kg bw/day.

In total 10 females from control, 11 females each from 30 and 100 mg/kg bw/day, 12 females from 300 mg/kg bw/day and 3 females from 1000 mg/kg bw/day dose groups gave birth to live litters and successfully reared young to Day 5 of age. At 1000 mg/kg bw/day data from pregnant females that failed to litter and females that showed total litter loss post partum were also taken into consideration.

There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries. Although fewer Group 5 animals showed implantation sites reflecting the in-life data there was no evidence that the non-pregnant or non-lactating animals were not cycling normally.

Dose descriptor:

NOEL

Remarks:

(reproductive performance)

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: absence of effect at 300 mg/kg

Dose descriptor:

NOAEL

Remarks:

(parental toxicity)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See below results for additional details. Mean litter weights were lower 1000 mg/kg bw/day reflecting the lower litter size.

Food efficiency:

not examined

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):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Offspring Litter Size, Sex Ratio and Viability
At 1000 mg/kg bw/day the mean number of corpora lutea was lower than control, however considering the small group size, this probably reflect normal biological variation and, at this stage, an association with treatment is not considered proven. The mean number of implantation sites was also lower than control but this mainly reflected the previous low corpora lutea count and, while higher mean pre-implantation loss was observed this was mainly due to one litter. However there was a clear increase in mean post-implantation loss with a corresponding reduction in live litter size on Day 1. This finding has to be viewed in the context of a further three females that either lost their litter in utero or very shortly after delivery. Overall offspring viability of the litters reared to weaning was lower than control and this value excludes two litters that both showed total litter loss post partum. It should be noted that many dams at this dosage showed excessively long gestation length and this prolonged gestation length would have been expected to compromise the offspring at parturition and lead to lower post partum survival, particularly during the early lactation period assessed as part of this study. Sex ratio of the offspring appeared unaffected by maternal treatment indicating that there was no selective effect on survival for either sex.

There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30, 100 or 300 mg/kg bw/day. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

Offspring Growth and Development
At 1000 mg/kg bw/day there was no adverse effect of treatment on the initial body weight of the offspring on Day 1 or subsequent body weight gain to Day 4. It could be argued that given the longer gestation period and smaller size for these litters, a higher offspring body weight at Day 1 may have been expected. It should also be noted that two litters showed total litter loss and these “inferior” litters have not been included in the mean calculation which is based on a very small group size. Mean litter weights were lower at this dosage reflecting the lower litter size.

There was considered to be no adverse effect of treatment on offspring body weight on Day 1 and subsequent body weight gain to Day 4 at 30, 100 or 300 mg/kg bw/day.

Clinical signs apparent for offspring were typical of the age and neither the incidence or distribution indicated any adverse effect of treatment at 30, 100 or 1000 mg/kg bw/day although the number of offspring assessed at the high dosage was much lower than the other groups on the study.

For all dosages the success rate at assessment of surface righting for the offspring at Day 1 of age was lower than control, however there was no dosage relationship and these differences probably reflect normal biological variation rather than any treatment related effect.

Necropsy - Offspring
Necropsy findings apparent for offspring were typical for the age observed and the low incidence and distribution of these observations did not indicate any effect of maternal treatment at 30, 100, 300 or 1000 mg/kg bw/day.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Including the survival, growth and development of the offspring

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

The No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring, was considered to be 300 mg/kg bw/day.

Executive summary:

The test item was administered by gavage to four groups, each of twelve male and twelve female Wistar rats, for up to eight weeks, including a two week prepairing phase, pairing, gestation and early lactation (Days 1 through 4 post partum) for females, at dose levels of 30, 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.
At 1000 mg/kg bw/day, there was no effect on mating (all 12 females mated), but the subsequent pregnancy rate was poor with three females failing to achieve pregnancy. Three of the nine pregnant females were not observed to give birth to a litter and one pregnant female was killed around the time of parturition. Two of the remaining females showed total litter loss post partum and only three females successfully reared their young to Day 5 of lactation. There was no effect of treatment on mating and fertility at 30, 100 or 300 mg/kg bw/day.
At 1000 mg/kg bw/day, there was a clear increase in the length of gestation for the five females observed to give birth to a litter, with a mean gestation length of 24.9 days for the high dose compared to 22.5 days for the control group. There was no effect of treatment on gestation length at 30, 100 or 300 mg/kg bw/day.
At 1000 mg/kg bw/day, assessment of any effect of treatment on corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age was frustrated by the small group size, although a clear increase in post implantation loss was apparent and supported by the incidence of post partum total litter loss observed at this dosage.
There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30, 100 or 300 mg/kg bw/day.
At 1000 mg/kg bw/day, assessment of any effect of treatment on offspring growth and development was frustrated by the small group size. There were, however, no adverse effects of treatment observed on pup clinical signs or pup body weights on post partum (lactation) Days 1 and 4.
Offspring body weight and body weight gain, surface righting ability on Day 1, clinical signs and necropsy findings did not indicate any effect of treatment at 30, 100 or 300 mg/kg bw/day.
Within the confines of this study, the No Observed Adverse Effect Level (NOAEL) for toxicity was considered to be 100 mg/kg bw/day. The No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring, was considered to be 300 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is considered to be reliable with a klimisch score of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined 28 -day repeated toxicity and reproduction screening study (Envigo, 2016):
The test item was administered by gavage to four groups, each of twelve male and twelve female Wistar rats, for up to eight weeks, including a two week prepairing phase, pairing, gestation and early lactation (Days 1 through 4 post partum) for females, at dose levels of 30, 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.
At 1000 mg/kg bw/day, there was no effect on mating (all 12 females mated), but the subsequent pregnancy rate was poor with three females failing to achieve pregnancy. Three of the nine pregnant females were not observed to give birth to a litter and one pregnant female was killed around the time of parturition. Two of the remaining females showed total litter loss post partum and only three females successfully reared their young to Day 5 of lactation. There was no effect of treatment on mating and fertility at 30, 100 or 300 mg/kg bw/day.
At 1000 mg/kg bw/day, there was a clear increase in the length of gestation for the five females observed to give birth to a litter, with a mean gestation length of 24.9 days for the high dose compared to 22.5 days for the control group. There was no effect of treatment on gestation length at 30, 100 or 300 mg/kg bw/day.
At 1000 mg/kg bw/day, assessment of any effect of treatment on corpora lutea count, preimplantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age was frustrated by the small group size, although a clear increase in post implantation loss was apparent and supported by the incidence of post partum total litter loss observed at this dosage.
There was considered to be no effect of treatment on the corpora lutea count, pre-implantation loss, numbers of implantations, post-implantation loss, litter size and sex ratio at birth/Day 1 and subsequent offspring survival to Day 4 of age at 30, 100 or 300 mg/kg bw/day.
At 1000 mg/kg bw/day, assessment of any effect of treatment on offspring growth and development was frustrated by the small group size. There were, however, no adverse effects of treatment observed on pup clinical signs or pup body weights on post partum (lactation) Days 1 and 4.
Offspring body weight and body weight gain, surface righting ability on Day 1, clinical signs and necropsy findings did not indicate any effect of treatment at 30, 100 or 300 mg/kg bw/day.
Within the confines of this study, the No Observed Adverse Effect Level (NOAEL) for toxicity was considered to be 100 mg/kg bw/day. The No Observed Effect Level (NOEL) for reproduction, including the survival, growth and development of the offspring, was considered to be 300 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study in time mated female Wistar Han rats was conducted using the registered substance.
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.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

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.

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).

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.

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.

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

Abnormalities:

not specified

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)

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

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

visceral/soft tissue: urinary

Description (incidence and severity):

Se developmental results section

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

 

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

 

 

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and was conducted in accordance with a relevant OECD Guideline

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

Prenatal Developmental Toxicity study in rats (CRL, 2023)
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.

Justification for classification or non-classification

Based on the available data, no classification for reproduction toxicity is required for 5,5'-Dithiodi-1,3,4-thiadiazole-2(3H)-thione according to the Regulation EC°1272/2008.