Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to Reproduction (screening): oral: gavage, rat (Han Wistar) m/f, 10/sex/dose, 0, 50, 150, 500 mg/kg bw/d(nominal) in PEG 400, (OECD TG 422, GLP): no adverse effects in reproductive performance or in offspring noted, NOAEL = 24 mg/kg (systemic toxicity), NOAEL = 500 mg/kg (reproductive toxicity), not toxic to reproduction

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-12 - 2017-10-11 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD 422 guideline for testing of chemicals adopted 29 July 2016: Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 to 8°C).
Species:
rat
Strain:
Wistar
Remarks:
RccHan™;WIST
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar (RccHan™;WIST) was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Envigo RMS (UK).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Males 78 to 84 days old. Females 78 to 84 days old
- Weight at study initiation:
Males 308 to 360 g, Females 192 to 244 g (start of treatment)
- Fasting period before study:
no
- Housing:
Rodent facility: Limited barrier - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods.
Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage:
Pre-pairing: up to five animals of one sex
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter
Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.
- Diet (e.g. ad libitum):
SDS VRF1 Certified pelleted, non-restricted (removed overnight before blood sampling for hematology, blood chemistry and thyroid hormones investigations).
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water (e.g. ad libitum):
Potable water from the public supply via polycarbonate bottles with sipper tubes ad libitum. Bottles were changed at appropriate intervals.
- Acclimation period:
Males: eight days before commencement of treatment.
Females: 22 days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
Monitored and maintained within the range of 20-24ºC.
- Humidity (%):
Monitored and maintained within the range of 40-70%.
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr):
Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light):
Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES: From: arrival To: necropsy
Males: 26 April 2017 - 9 June 2017
Females: 12 April 2017 - 23 to 26 June 2017
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Method of preparation : The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation:Weekly.
Storage of formulation: Refrigerated (2 to 8°C) for up to fifteen days.
Ambient (15 to 25°C) for up to 24 hours.
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

- VEHICLE
- Concentration in vehicle: 0, 20, 60, 200 mg/ml)
- Amount of vehicle (if gavage): 2.5 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability and homogeneity:Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 of treatment (for each group) and on Day 12 of lactation (females) were analyzed for achieved concentration of the test item.
The Week 1 analysis revealed that Group 2 and 3 achieved concentrations were low (40.0 and 21.3% of nominal). Additional samples were taken in Week 1 for confirmatory an analysis to ensure the robustness of the analytical technique. The preparation intended for use in Week 2 prepared for Groups 2 and 3 were used for dosing the reminder of Week 1.
Duration of treatment / exposure:
Two weeks before pairing to necropsy (males: Week 5 (36 days), females: Day 14 past partum (51-54 days))
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Analyzed mean concentrations of group 3 was lower than nominal concentration ranging from -51.9% to -15.5% at 20 mg/mL, corresponding to a dose of 24 mg/kg/day.
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Analyzed mean concentration of group 2 was lower than nominal concentration ranging from -24.5% to -8.0% at 60 mg/mL.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
The mean concentrations of Group 4 at 200 mg/mL were within applied limits +10/-15%, confirming the accuracy of formulation.
No. of animals per sex per dose:
10/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels of 50, 150 and 500 mg/kg/day were selected after consultation with the Sponsor following the completion of the preliminary study. In that study there were no effects on clinical condition, water consumption (visual) or macropathology. There was a dose dependent effect on body weight loss with males and females that received 500 and 750 mg/kg/day showing a marked mean body weight loss following initial treatments (Days 1 to 4 - males 12 g or 37 g; females 4 g and 12 g, respectively) which corresponded to the low food consumption also seen at these dose levels (Days 1 to 4). Males receiving 750 mg/kg/day showed an overall loss in body weight of 11 g. Mean female spleen weights were high at 750 mg/kg/day. No such observations were made at 250 mg/kg/day.
Therefore, the high dose level for this study was 500 mg/kg/day with the intermediate and low dose levels chosen to allow the determination of a dose response.

- Rationale for animal assignment (if not random):
On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days evaluation, animals that failed to exhibit 4-5 day cycles are not allocated to the study.
On Day 1 of study all animals were weighed and body weights were reviewed Study Management before dosing commenced by to ensure variations in body weight of animals did not exceed +/- 20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before allocation: Irregular estrous cycle (One female)
Positive control:
not required
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males: Week 1 - daily; Week 2 onwards - once each week
F0 females: Week 1 - daily; Week 2 - once; Gestation phase - Days 0, 7, 14 and 20; Lactation phase - Days 1, 6 and 12
Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
At completion of dosing each group
One to two hours after completion of dosing
As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males:
Weekly during acclimatization
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.
F0 females:
Weekly during acclimatization
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7 and 13 of lactation.
On the day of necropsy.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Blood samples were collected after overnight withdrawal of food at the following occasion: At termination: The five lowest numbered surviving males and the first five lactating females with a surviving litter per group.
- Anaesthetic used for blood collection: Yes. Animals were held under light general anesthesia induced by isoflurane.
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters below were examined: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
* Derived value calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. In the presence of platelet clumping a manual count of the differential white blood cell parameters was performed.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood samples were collected after overnight withdrawal of food at the following occasion: At termination: The five lowest numbered surviving males and the first five lactating females with a surviving litter per group.
- Animals fasted: Yes
- How many animals: 5/sex/group
- Parameters below were examined:
Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal.
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and the first five lactating females in each group was measured (before dosing)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations:
Blood samples were collected as follows: At termination: All surviving adult males and females (no samples were obtained from animals which fail to litter or with a total litter loss).
- How many animals: all
- Dose groups that were examined: all
- Parameters examined: Thyroid Hormone Analysis
Oestrous cyclicity (parental animals):
Dry and wet smears were taken as follows:
Dry smears:For 15 days before pairing using cotton swabs.
Wet smears: Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating.
For four days before scheduled termination (nominally Days 11-14 of lactation).
Sperm parameters (parental animals):
Parameters examined in the male parental generation: testis weight, epididymis weight
For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
Litter observations:
STANDARDISATION OF LITTERS: No

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, anogenital distance (AGD), presence of nipples/areolae in male pups

Parturition Observations and Gestation Length
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Records Made During Littering Phase
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Thyroid Hormone Analysis: Blood samples were collected as follows:
Day 4 of age F1 offspring, two females per litter (where possible ensuring that the number of female offspring did not fall below three).
No pups were allocated to these procedures if the resultant live litter size would fall below eight pups/litter.
- one for T4 (serum)#
- one for TSH (plasma)
# priority given to serum sample
Day 13 of age:F1 offspring, two males and two females per litter (where possible).
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes, see table in "Any other information on materials and methods"

All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of Necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to mate: Day 25 after last day of pairing.
F0 females failing to produce a viable litter: If an estrus smear was seen following completion of the pairing period animals would have been terminated as soon as logistically possible.
If no estrus smear is seen, animals would have been terminated on Day 25 after last day of pairing.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 14 of lactation.


HISTOPATHOLOGY: Yes, see table in "Any other information on materials and methods"
Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All animals killed prematurely.
The five lowest numbered surviving F0 males and five lowest numbered lactating F0 females in Groups 1 and 4 at scheduled termination.
Kidneys and Stomach: The five lowest numbered surviving F0 males and five lowest numbered lactating F0 females in Groups 2 and 3 at scheduled termination.
Abnormalities only: All remaining F0 animals.
Routine staining: Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy
Tissues preserved for examination were examined.
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Postmortem examinations (offspring):
SACRIFICE
Selected offspring for thyroid hormone analysis – Day 4 of age.
Scheduled kill – Day 13 of age
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
F1 offspring on Day 4 of age: Blood sampling required.
Externally normal offspring discarded without examination.
Externally abnormal offspring examined, and retained pending possible future examination.
F1 offspring on Day 13 of age: Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination
Thyroid glands were preserved from one male and one female in each litter.
Statistics:
Due to limitations of this free-text field, see "Any other information on materials and methods"
Reproductive indices:
Estrous Cycle
The percentage females showing the following classifications of estrous cycles before treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination

Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating / Animals paired) * 100
Conception rate (%) = (Number of animals achieving pregnancy / Animals mated) * 100
Fertility index (%) = (Number of animals achieving pregnancy / Animals paired) * 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (Number of live litters born / Number pregnant) * 100
Offspring viability indices:
Litter Size
Individual litter values were tabulated for the number of implantation sites, total at Day 1 and live at Days 1, 4 (before and after blood sampling) and 13 of age. Group mean litter size and SD were calculated from the individual litter values.

Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) * 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) * 100
Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) * 100
Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) * 100
Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.
Percentage males = (Number of males in litter / Total number of offspring in litter) * 100
Group mean values were calculated from individual litter values.

Offspring Examinations
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.
A check was performed to assess for the presence or absence of nipple/areolae for the male offspring. As no nipples were present, no data are included.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Post-dose observations included chin rubbing and salivation and were observed immediately after administration in the majority of cases, at 150 and 500 mg/kg/day and continued through the gestation and lactation phases for females at 150 and 500 mg/kg/day. Rales was evident on days 2, 5 and 6 of treatment in both males and females at 500 mg/kg/day. Two males at 500 mg/kg/day had loose faeces.
Clinical signs observed at routine examination comprised of rales in 2/10 males and 6/10 females at 500 mg/kg/day before pairing. Rales was also evident after mating in one female at 500 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Four animals were either killed for welfare reasons or found dead during the treatment phase.
Male animal No.6, who received 500 mg/kg/day, was found dead on Day 33 of treatment. There were no clinical signs relating to treatment, although rales was observed 1-2 post-dose on Day 5 of treatment. The macroscopic examination revealed a distended stomach and red fluid in the thorax with marked alveolar exudate seen microscopically. This was thought to be most likely related to the dosing procedure. Cortical tubular pigment, which was considered to be related to treatment, was seen in the kidney of this individual.
At 500 mg/kg/day female, No. 46, was killed for welfare reasons on Day 6 of treatment. On Day 5 of treatment at the 1-2 hours post-dose and as late as possible in the working day observations, signs of rales was evident which continued into Day 6 accompanied with gasping. Dark coloured depressions in the corpus of the stomach, seen macroscopically, correlated with mucosal erosion/ulceration and necrosis which was considered to be the major factor contributing to death. The stomach findings were similar to those seen in terminal animals and were considered to be treatment related.
One Control female (No. 63) was killed for welfare reasons on Day 11 of treatment. Clinical signs included piloerection, a thin build, hunched posture and underactive. There were no macroscopic findings attributed to dosing, however moderate multifocal necrosis which was seen in the brain on microscopic examination was the major factor for death in this individual.
One Group 3 female (No. 71), at 150 mg/kg/day, was found dead on Day 15 of treatment. The animal was seen to have convulsed prior to dosing. There were no other clinical signs and no post-dose observations for this animal however clear fluid was found in the pharynx and trachea at macroscopic examination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males receiving 500 mg/kg/day showed marked mean bodyweight loss following initial treatments over Week 0 to 1 (16 g). Thereafter from Week 1 onwards, males receiving 500 mg/kg/day showed body weight gains with mean body weight gains being higher than controls during Week 1 to 3 but lower during Weeks 3-5. The overall bodyweight change from Week 0 to Week 5 in males receiving 500 mg/kg/day was 53 % lower than Control. There was no effect on bodyweight at 50 or 150 mg/kg/day in males and females or 500 mg/kg/day in females during the treatment phase.
There was no effect on bodyweight at 50 or 150 mg/kg/day in males and females or 500 mg/kg/day in females during the treatment phase.
During gestation, Group 4 females receiving 500 mg/kg/day persistently showed slightly lower bodyweights than Controls. The overall weight gain from Day 0-20 was 22.7% lower than Controls, reflecting low weight gain during Days 0-7 and 14-20.
Bodyweights during gestation were unaffected at 50 and 150 mg/kg/day.
In the lactation phase, low bodyweights continued in females receiving 500 mg/kg/day, however, the overall bodyweight gain from Day 1 to 13 was comparable with Control.
Bodyweights during lactation were unaffected at 50 and 150 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of males and females receiving 500 mg/kg/day was markedly low when compared to Control during Week 1 (males: 59%; females 85%). Food intake of these animals increased during Week 2, but remained lower than controls in males for the duration of the treatment period. The overall mean food consumption for males receiving 500 mg/kg/day was 82% of Control.
Food consumption at 50 and 150 mg/kg/day was unaffected by treatment.
Food consumption was unaffected at all treatment levels during the gestation phase.
During the lactation phase, females receiving 500 mg/kg/day had low food consumption when compared with Control. The mean food consumption for days
1-12 of lactation was 81% of Control.
Food consumption was unaffected at 50 and 150 mg/kg/day in the lactation phase.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The haematological examination of peripheral blood performed after five weeks of treatment for males and on Day 14 of lactation for females did not reveal any toxicologically significant differences from control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the slightly low hematocrit in males receiving 500 mg/kg/day, high activated partial thromboplastin time in males at 50 and 150 mg/kg/day, high reticulocyte count and low mean cell hemoglobin concentration in females receiving 500 mg/kg/day and high monocyte count in females at 50 and 500 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma performed after five weeks of treatment for males and on Day 14 of lactation for females indicated, when compared to the controls, a marked decrease in alanine aminotransferase concentration in males and females at 50, 150 and 500 mg/kg/day, with a dose-dependent response (36%, 25% and 14% of Control for males respectively; 48%, 27% and 16% of Control for females respectively).
Alkaline phosphatase (at 500 or 150 mg/kg/day), glucose values and plasma phosphorous levels in males were low, and attained statistical significance, with a dose dependent trend. No similar finding was observed in the females.
Total protein and globulin values were low at all treatment levels with statistical significance obtained at 500 mg/kg/day in males. The album/globulin ratio was also statistically significantly increased at all treatment levels.
All other differences from controls, including those that attained statistical significance, were minor, lacked dose-relationship or were confined to one sex or showed the opposite direction of effect between the sexes and were therefore attributed to normal biological variation. Such differences included the variations of bile acid concentration in both sexes, the non-dose dependent increases in calcium in females, the low calcium and potassium values in males at 500 mg/kg/day and the high potassium values in females at 500 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory reactivity responses were unaffected by treatment.
Females receiving 500 mg/kg/day showed low grip strength scores, with hindlimb grip strength attaining statistical significance (p<0.05).
Motor activity was unaffected by treatment.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The level of T4 was low in males receiving 500 mg/kg/day.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, the adjusted mean thyroid weight was statistically significantly high in males treated at 500 mg/kg/day.
All other organ weights were unaffected by treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Dark area(s) of the stomach were observed in both sexes receiving 500 mg/kg/day. Depressions in the corpus were seen in a male receiving 500 mg/kg/day and females receiving 150 mg/kg/day. A single incidence of a thickened corpus was seen in a male receiving 150 mg/kg/day with a thickened limiting ridge also recorded in a male receiving 500 mg/kg/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with 2,5-Dimercapto-1,3,4-thiadiazole were seen in the kidney and stomach.
Kidney: An increase incidence of cortical tubular pigment was seen in both sexes receiving 500 mg/kg/day and males receiving 150 mg/kg/day.
Special stains, consisting of Perls’ stain and Schmorl’s stains, were performed on the kidneys of four animals comprising a control and a high dose animal of each sex. The pigment was negative for Perls’ and positive for Schmorl’s, indicating that the pigment was lipofuscin.
Stomach: Findings were seen in the stomach of both sexes receiving 150 and 500 mg/kg/day.
The findings were seen mostly in the glandular region and comprised mucosal erosion/ ulceration, haemorrhage and oedema in the lamina propria, mucosal regeneration and submucosal inflammatory cells. In addition females receiving 500 mg/kg/day also had epithelial hyperplasia affecting the non-glandular stomach and limiting ridge.
Incidental Findings: All other findings were considered to be incidental and unrelated to treatment.
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Estrous cyclicity, pre-coital interval, mating performance, fertility and gestation index were considered unaffected by treatment with 2,5-Dimercapto-1,3,4-thiadiazole.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No histopathological changes in the testes or examined sperm parameters were noted.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cyclicity, pre-coital interval, mating performance, fertility and gestation index were considered unaffected by treatment with 2,5-Dimercapto-1,3,4-thiadiazole.
A statistically significant shift in gestation length was observed in the 500 mg/kg/day group with only one female showing a 22 or 22.5 day gestation length and more showing a 23 or 23.5 day gestation length, compared with Controls and the historical control data range.
All females showed diestrus at termination.
Key result
Dose descriptor:
NOAEL
Effect level:
24 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No signs were recorded that were considered to be related to parental treatment with 2,5-Dimercapto-1,3,4-thiadiazole.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Litter size, offspring survival to Day 13 of age and sex ratio were unaffected by parental treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweights and bodyweight gain were low for male offspring on Day 4, 7 and 13 of age at 150 and 500 mg/kg/day and for female offspring on Day 7 and 13 of age at 150 and 500 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Description (incidence and severity):
Only thyroid hormone analysis performed. There was no effect of treatment on the circulating levels of thyroxine (T4) in the offspring on Day 13 of age
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
There was no effect of parental treatment upon ano-genital distances in male offspring, however in female offspring a statistically significant dose-dependent decrease in the adjusted ano-genital distances was observed, however this direction of difference is considered to be of no toxicological significance.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination of offspring revealed no test item related lesions.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the circulating levels of thyroxine (T4) in the offspring on Day 13 of age.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
F1 generation was not dosed, test item was possibly received indirect via the dams
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
gross pathology
developmental immunotoxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no
Conclusions:
It was concluded that the oral administration of 2,5-Dimercapto-1,3,4-thiadiazole to parental Han Wistar rats at dose levels of 50, 150 or 500 mg/kg/day administered for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation caused low bodyweight and food consumption effects at 500 mg/kg/day and histopathological findings in the stomach which contributed to the death of one female at 500 mg/kg/day and at 150 mg/kg/day. The no-observed adverse-effect level (NOAEL) of 2,5-Dimercapto-1,3,4-thiadiazole for toxicity was considered to be 24 mg/kg/day when adjusted for the results of the formulation analysis.
Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, 2,5-Dimercapto-1,3,4-thiadiazole showed no conclusive evidence of being an endocrine disruptor and therefore the no-observed adverse-effect level (NOAEL) for reproductive/developmental toxicity was considered to be 500 mg/kg/day.
Executive summary:

The purpose of this study according to OECD 422 under GLP was the assessment of general systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of 2,5‑Dimercapto‑1,3,4‑thiadiazole by oral gavage administration for at least five weeks.

Three groups of ten male and ten female Han Wistar rats received 2,5‑Dimercapto‑1,3,4‑thiadiazole at doses of 50, 150 or 500 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, PEG 400, at the same volume dose as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

Results

F0 responses

The homogeneity and stability was confirmed for 2,5-Dimercapto-1,3,4-Thiadiazole in PEG 400 formulations at nominal concentrations of 1 mg/mL and 400 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. 

The mean concentrations of Group 4 were within applied limits +10/-15%, confirming the accuracy of formulation. Groups 2 and 3 were lower than nominal concentration ranging from -52% to -15.5%.

Four animals were either killed for welfare reasons or found dead during the treatment phase, however only the following death was considered to be related to treatment.

At 500 mg/kg/day female, No. 46, was killed for welfare reasons on Day 6 of treatment. On Day 5 of treatment at the 1-2 hours post-dose and as late as possible in the working day observations, signs of rales was evident which continued into Day 6 accompanied with gasping. Dark coloured depressions in the corpus of the stomach, seen macroscopically, correlated with mucosal erosion/ulceration and necrosis which was considered to be the major factor contributing to death. The stomach findings were similar to those seen in terminal animals and were considered to be treatment related.

Post-dose observations included chin rubbing and salivation and were observed immediately after administration in the majority of cases, at 150 and 500 mg/kg/day and continued through the gestation and lactation phases for females at 150 and 500 mg/kg/day. Rales was evident on days 2, 5 and 6 of treatment in both males and females at 500 mg/kg/day. Two males at 500 mg/kg/day had loose faeces.

Males receiving 500 mg/kg/day showed marked mean bodyweight loss following initial treatments over Week 0 to 1 (16 g). Thereafter from Week 1 onwards, males receiving 500 mg/kg/day showed body weight gains with mean body weight gains being higher than controls during Week 1 to 3 but lower during Weeks 3-5. The overall bodyweight change from Week 0 to Week 5 in males receiving 500 mg/kg/day was 53 % lower than Control. There was no effect on bodyweight at 50 or 150 mg/kg/day in males and females or 500 mg/kg/day in females during the treatment phase.

During gestation, Group 4 females receiving 500 mg/kg/day persistently showed lower bodyweights than Controls. The overall weight gain from Day 0-20 was 22.7% lower than Controls. Bodyweights during gestation were unaffected at 50 and 150 mg/kg/day.

In the lactation phase, low bodyweights continued in females receiving 500 mg/kg/day, however, the overall bodyweight gain from Day 1 to 13 was comparable with Control. Bodyweights during lactation were unaffected at 50 and 150 mg/kg/day.

Food consumption of males and females receiving 500 mg/kg/day was markedly low when compared to Control during Week 1 (males: 59%; females 85%). Food intake of these animals increased during Week 2, but remained lower than controls in males for the duration of the treatment period. The overall mean food consumption for males receiving 500 mg/kg/day was 82% of Control. Food consumption at 50 and 150 mg/kg/day was unaffected by treatment.

Food consumption was unaffected at all treatment levels during the gestation phase.

During the lactation phase, females receiving 500 mg/kg/day had low food consumption when compared with Control. The mean food consumption for days 1-12 of lactation was 81% of Control. Food consumption was unaffected at 50 and 150 mg/kg/day in the lactation phase.

Dark area(s) of the stomach were observed in both sexes receiving 500 mg/kg/day. Depressions in the corpus were seen in a male receiving 500 mg/kg/day and females receiving 150 mg/kg/day. A single incidence of a thickened corpus was seen in a male receiving 150 mg/kg/day with a thickened limiting ridge also recorded in a male receiving 500 mg/kg/day.

Microscopic changes related to treatment with 2,5-Dimercapto-1,3,4-thiadiazole were seen in the kidney and stomach.

An increase incidence of cortical tubular pigment was seen in both sexes receiving 500 mg/kg/day and males receiving 150 mg/kg/day. Special stains, consisting of Perls’ stain and Schmorl’s stains, were performed on the kidneys of four animals comprising a control and a high dose animal of each sex. The pigment was negative for Perls’ and positive for Schmorl’s, indicating that the pigment was lipofuscin.

Findings were seen in the stomach of both sexes receiving 150 and 500 mg/kg/day. The findings were seen mostly in the glandular region and comprised mucosal erosion/ ulceration, haemorrhage and oedema in the lamina propria, mucosal regeneration and submucosal inflammatory cells. In addition females receiving 500 mg/kg/day also had epithelial hyperplasia affecting the non-glandular stomach and limiting ridge.

Circulating T4 levels were low in males receiving 500 mg/kg/day and mean adjusted thyroid weight was marginally but statistically significantly high, but there were no supporting microscopic pathology changes in the thyroids.

 

F1 responses

The clinical condition, litter size, sex ratio, survival indices and body weight gain of offspring were unaffected by parental treatment.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

The ano-gential distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to paternal treatment were recorded.

 

Conclusion

It was concluded that the oral administration of 2,5-Dimercapto-1,3,4-thiadiazole to parental Han Wistar rats at dose levels of 50, 150 or 500 mg/kg/day administered for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation caused low bodyweight and food consumption effects at 500 mg/kg/day and histopathological findings in the stomach which contributed to the death of one female at 500 mg/kg/day and at 150 mg/kg/day. The no-observed adverse-effect level (NOAEL) of 2,5-Dimercapto-1,3,4-thiadiazole for toxicity was considered to be 24 mg/kg/day when adjusted for the results of the formulation analysis.

Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, 2,5‑Dimercapto‑1,3,4‑thiadiazoleshowed no conclusive evidence of being an endocrine disruptor and therefore the no-observed adverse-effect level (NOAEL) for reproductive/developmental toxicity was considered to be 500 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Study was conducted on the registered substance itself acc. OECD TG 422 under GLP. Hence, the tonnage-driven data requirements under REACH are fully met, and the database is of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

See above, Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422), no adverse effects in reproductive performance or in offspring noted.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Based on lack of any adverse effects on reproductive toxicity (and general systemic toxicity), it is rather impossible to hypothesize a concrete mode of action.

This study included a screen for reproductive/developmental effects and the results obtained were unremarkable. Estrous cycles, pre-coital interval, mating performance, fertility, litter size and gestation index were unaffected by treatment. A statistically significant shift in gestation length was observed at 500 mg/kg/day only one female showing a 22 or 22.5 day gestation length and more showing a 23 or 23.5 day. The clinical condition of the offspring, their survival, growth, sex ratio, ano-genital distance in male offspring on Day 1 of age and male nipple counts on Day 13 of age showed no adverse effects of parental treatment. Female offspring revealed a statistically significant dose-dependent decrease in the adjusted ano-genital distances. There were also no signs in the small number of decedent offspring, or offspring at termination on Day 13 of age that were considered to be related to parental treatment.

The oral administration of the test item lead to a NOAEL = 24 mg/kg bw/day for systemic toxicity, whereas the NOAEL for reproductive toxicity was with 500 mg/kg/day equal to the highest dose tested, which already lead to deaths in the parental generation. So further any effects on reproductive performance as secondary effects of maternal treatment can be excluded.

No definitive human relevance framework can be described due to the lack of any effects on reproductive performance securing any postulation, and no conclusion on biological plausibility can be drawn.

Despite the fact that no mode of action analysis can be performed, no data gap was identified here. The tonnage-driven data requirements under REACH were fully met, and the lack of relevance of the observed effects does also not indicate any high hazard for humans and so does not trigger any further examinations.

Justification for classification or non-classification

On the basis of the results of the reproduction/developmental toxicity screening test of DMTD on rats, the no observed adverse effect level (NOAEL) of the test item was determined. The oral (gavage) administration of the test item to Han Wistar rats, at dose levels of 50, 150 or 500 mg/kg bw/day (nominal) did not produce any adverse effects in reproductive performance or in offspring.

The oral administration of the test item lead to a NOAEL = 24 mg/kg bw/day for systemic toxicity, whereas the NOAEL for reproductive toxicity was with 500 mg/kg/day equal to the highest dose tested, which already lead to deaths in the parental generation.

 So, the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day as well as the NOAEL for reproductive toxicity was considered to be 1000 mg/kg bw/day within the confines of this study.

According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, a substance must be considered as reproductive toxicant under the following conditions: Suspected human reproductive toxicant: Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification. Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.

No effects on fertility were noted up to 500 mg/kg, systemic toxicity was noted already at 24 mg/kg bw/d, so secondary effects con further be excluded. Consequently the criteria for classification as reproductive toxicant (Cat. 2) are not met, the substance does not need to be classified according to Regulation 1272/2008.