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 Han™:RccHan™:WIST strain 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:

subchronic

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 (Fulcher 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 developmental toxicity study in rat is proposed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available (further information necessary)

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

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.