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Administrative data

Description of key information

Repeated Dose toxicity: Subacute / reproductive toxicity study 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): NOAEL = 24 mg/kg bw/d (analytical), based on 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. No specific target organ toxicity was identified, no classification as STOT RE Cat. 2 was triggered.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
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
Details on route of administration:
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth
Vehicle:
polyethylene glycol
Remarks:
PEG 400
Details on oral 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 weighing’s 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
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.
For further details, see attachment
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
Observations and examinations performed and frequency:
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
Sacrifice and pathology:
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.
Other examinations:
See Reproductive Toxicity section
Statistics:
Due to limitations of this free-text field, see "Any other information on materials and methods"
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.
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
Other effects:
no effects observed
Description (incidence and severity):
See subchapter "reproductive toxicity"
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
Critical effects observed:
no

See subchapter "reproductive toxicity"

Conclusions:
The study was performed under GLP according to OECD TG 422 without deviations. Hence, the results can be considered sufficiently reliable to assess the potential repeated dose toxicity (and reproductive toxicity) of the test item, especially taking into account the prolonged exposure duration (minimum 5 weeks) compared to a regular subacute 28-day study and the fact that pregnant animals are in general more susceptible for toxic effects.
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. No evidence for classification as STOT RE was given as the only relevant finding in the stomach was considered a local, no systemic effect.
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-thiadiazolefor 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
24 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.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

A mode of action analysis in its classic sense cannot be performed for repeated dose toxicity as there is only one repeated dose study available in one species, inclusive reproductive screening. However, the rat is an established model for human risk assessment, interspecies differences are well studied and allow transfer conclusions to humans for most chemicals exhibiting certain effects in the rat. Nevertheless it is aimed to follow the WHO IPCS template as far as possible:

 

Problem formulation

The present MoAA aims to describe the nature of the observed effects with regard to repeated dose systemic toxicity aims to draw conclusions on their relevance for humans.

 

Hypothesised Mode of action Statement

There were some effects noted in various organs and a general poorer health state is associated. A dose-response was observed, the degree of severity increases with the dosage. At the lowest dose, no relevant effects were noted. With increasing doses, the effects may be more interpreted as adverse. Besides local irritation in the stomach, all effects were judged not consistent, and not to allow to conclude a specific organ toxicity.

 

Summary of data for use in Mode of Action Analysis

In the available OECD 422 on the registered substance itself (oral: gavage, Han Wistar rat m/f, 10/sex/dose, doses: 0, 50, 150, 500 mg/kg bw/d (nominal), duration: subacute), the NOAEL was delineated as 24 mg/kg bw/d (analytical).

The oral administration of 2,5-Dimercapto-1,3,4-thiadiazole to parental Han Wistar rats was generally well tolerated. One female animal at 150 mg/kg/day and one male animal at 500 mg/kg/day were found dead during treatment and one Control female and one female at 500 mg/kg/day were euthanised for welfare reasons. It was considered that the only death attributable to treatment was female No. 46 at 500 mg/kg/day due to the mucosal erosion/ulceration and necrosis seen in the stomach.

Macropathology revealed dark areas and depressions of the stomach which correlated with mucosal erosion/ ulceration and hemorrhage in the lamina propria. In addition, oedema, mucosal regeneration and submucosal inflammatory cells were seen after histopathological assessment. These stomach changes at 500 or 150 mg/kg/day were considered to be adverse and to be due to the moderate local irritation of the test item, which further contributed to the death of one female at 500 mg/kg/day and at 150 mg/kg/day. This effect was not present in the 50 mg/kg/day group and was considered dose-dependent. Cortical tubular pigment was seen in the kidneys in both sexes receiving 500 mg/kg/day and males receiving 150 mg/kg/day, however this finding was not considered adverse as there were no nephrotoxic findings in the clinical pathology.

Overall bodyweight gains were low for males at 500 mg/kg/day after 5 weeks of treatment and overall bodyweight gains were low at 500 mg/kg/day during the gestation phase. Bodyweights remained low during the lactation phase; however the bodyweight gains were comparable with Control. Bodyweights were unaffected at 50 and 150 mg/kg/day throughout the study. Overall food consumption was low for males at 500 mg/kg/day and females before pairing and during lactation at 500 mg/kg/day when compared with Control. Food consumption at 50 and 150 mg/kg/day was unaffected throughout the study.

 

Listing of key events identified for a specific Mode of Action

 

See the table in the next subchapter for incidence and severity. In general the following events were noted:

 

Key Event 1

Death

Key Event 2

bodyweight loss / diminished body weight in males

Key Event 3

Lower food consumption

Key Event 4

Macroscopic dark area(s) of the stomach, depressions in the corpus

Key Event 5

Increase incidence of cortical tubular pigment

 

All other examined parameters were not different from control.

 

Bradford Hill Considerations for Weight of Evidence Analysis of available data/information for Mode of Action Analysis in experimental species

 

Dose Response Relationships and Temporal Association

Conclusions on temporal association cannot be drawn, as there is only one OECD 422 study available with a study duration of approx. 6-7 weeks.

 

Dose (mg/kg bw/d)

Key Event 1 (severity)

Key Event 2 (severity)

Key Event 3 (severity)

Key Event 4 (severity)

Key Event 5 (severity)

50

No treatment-related deaths

no effect on bodyweight

No effects

No effects

No effects

150

1 treatment-related death

no effect on bodyweight

No effects

- Depressions in the corpus in females

-Mucosal Erosion/Ulceration – Glandular Region (1M / 2F)

- Haemorrhage in Lamina Propria – Glandular Region (1M / 1F)

- increase incidence of cortical tubular pigment in males

500

1 treatment-related death

Males showed marked mean bodyweight loss following initial treatments over Week 0 to 1.

The overall bodyweight change from Week 0 to Week 5 in males receiving 500 mg/kg/day was 53 % lower than Control.

No effect on bodyweight in females.

- Food consumption markedly low when compared to Control during Week 1 (males: 59%; females 85%). The overall mean food consumption for males receiving 500 mg/kg/day was 82% of Control.

- The mean food consumption for days 1-12 of lactation was 81% of Control

- Dark area(s) of the stomach were observed in both sexes.

- Depressions in the corpus in a male

-Mucosal Erosion/Ulceration – Glandular Region (4M / 2F)

- Haemorrhage in Lamina Propria – Glandular Region (4M / 5F)

- Submucosal Inflammatory Cells – Glandular Region (3M / 1F)

- Oedema and Epithelial Hyperplasia

- increase incidence of cortical tubular pigment in both sexes

 

As postulated above, a dose-response was observed, the degree of severity increases with the dosage.

 

Consistency & Specificity – Biological Plausibility

The key events follow a dose-response-curve, as depicted above (Dose Response Relationships). Key events 1-3, i.e. death, bodyweight loss and diminished food consumption are general signs of overall toxicity, and cannot be attributed to a certain MoA. However, they could be explained by the effects noted in the stomach which lead to the poor health condition. All effects in the stomach can be explained by local, irritating effects on the stomach. The substance was classified as Eye Dam. 1, and, although not classified as skin irr., in the dermal acute toxicity test there were also some effects noted after 24h continuous exposure, such as crust formation, small superficial scattered scabs, scab lifting to reveal glossy skin, yellow staining of the treatment sites. So upon repeated exposure on the stomach mucosa, irritating effects occurred. With regard to cortical tubular pigment which was seen in the kidneys 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. This finding was not considered adverse as there were no nephrotoxic findings in the clinical pathology. Further, males seem to be more susceptible here than females, and although no information on alpha-2u-globulin is available, this may indicate that these effects are even less relevant for humans.

 

Qualitative and Quantitative human concordance

There is no indication given that the obtained results may not be relevant for humans, no species-specifity was obvious, besides a possible indication from the severity of kidney affects as stated above.

 

Other potential Modes of Action

None identified

 

Uncertainties/Inconsistencies and Identification of Data Gaps

None identified

 

Conclusions in relation to problem formulation

It was shown that the effects in the stomach can be attributed to local effects of DMTD, which are dose-dependent. Those effects are expected to occur similarly in humans. Similarly, a poorer health condition can be expected as a consequence. The occurrence of lipofuscin in the kidneys does not need to be regarded as relevant effect as there were no nephrotoxic findings in the clinical pathology showing consistency.

There is no concrete evidence given that the obtained results may not be relevant for humans, no species-specifity was obvious. However, as human exposure is most likely to be way below the dosages used in the animal study, it can be concluded that, if any responses are observed at all, they will be of non-adverse, adaptive nature only, as the local effects noted in the stomach seem to follow a threshold-MoA.

Additional information

Justification for classification or non-classification

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 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 was generally well tolerated. However, it resulted in reduced body weight gain and macroscopic and microscopic changes in the stomach in animals of either sex treated with 150 and 500 mg/kg bw/day.

Target organ toxicity (repeated exposure), as defined by the Regulation, “means specific, target organ toxicity arising from a repeated exposure to a substance or mixture. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed are included. … Classification for target organ toxicity (repeated exposure) identifies the substance as being a specific target organ toxicant and, as such, it may present a potential for adverse health effects in people who are exposed to it”. Furthermore, “substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement”, and “3.9.2.7. Effects considered to support classification for specific target organ toxicity following repeated exposure...

... (c) any consistent and significant adverse change in clinical biochemistry, haematology, or urinalysis parameters;”

But also: “3.9.2.8. Effects considered not to support classification for specific target organ toxicity following repeated exposure

3.9.2.8.1. It is recognised that effects may be seen in humans and/or animals that do not justify classification. Such effects include, but are not limited to: ...

... (b) small changes in clinical biochemistry, haematology or urinalysis parameters and/or transient effects, when such changes or effects are of doubtful or minimal toxicological importance; ...

... (d) adaptive responses that are not considered toxicologically relevant;”

According to Regulation 1272/2008, Table 3.9.3, Guidance values to assist in Category 2 classification are 10 < C100 mg/kg body weight/day derived from a 90 day repeated dose study. For a short-term study, with a safety factor of 3, the boundary value would increase to 300 mg/kg.

Based on the available information, a classification as STOT RE Cat. 2 is not considered necessary as will be outlined below.

The reduced body weight gain does not account for any specific organ, and so only the macroscopic and microscopic changes in the stomach in animals of either sex treated with 150 and 500 mg/kg bw/day need to be regarded.

Macropathology revealed dark areas and depressions of the stomach which correlated with mucosal erosion/ ulceration and hemorrhage in the lamina propria. In addition, oedema, mucosal regeneration and submucosal inflammatory cells were seen after histopathological assessment. These stomach changes at 500 or 150 mg/kg/day were considered to be adverse and to be due to the moderate local irritation of the test item. This effect was not present in the 50 mg/kg/day group and was considered dose-dependent

These findings represent an adverse effect of treatment they are considered to reflect local irritation rather than any adverse systemic toxicity of the test item. Further, the increased incidence of cortical tubular pigment in both sexes is with 500 mg/kg bw/d above the guidance value and human relevance is, although likely, not absolutely evident.

Summarizing, the observed effects do not suffice for and justify a classification of DMTD as STOT RE Cat. 2.