Molybdenum, bis(dibutylcarbamodithioato)di-μ-oxodioxodi-, sulfurized

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2017 to 06 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Molybdenum, bis(dibutylcarbamodithioato)di-μ-oxodioxodi-, sulfurized

EC Number:

270-180-5

EC Name:

Molybdenum, bis(dibutylcarbamodithioato)di-μ-oxodioxodi-, sulfurized

Cas Number:

68412-26-0

Molecular formula:

C18H36N2Mo2O2S4X2 Where X = O or S

IUPAC Name:

Molybdenum oxide sulfide dibutyldithiocarbamate complex

Test material form:

solid: particulate/powder

Details on test material:

- Identification: Molybdenum, bis (dibutylcarbamodithioato)
- CAS Number: 68412-26-0

Specific details on test material used for the study:

- CAS Registry Number: 68412-26-0
- Physical state/Appearance: Yellow Powder
- Purity: This substance has an Unknown or Variable composition, is a Complex reaction product, or a Biological material (UVCB)

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males 70 to 77 days old; females 84 to 91 days old.
- Weight at study initiation: Males 333 to 393 g; females 257 to 293 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals, ad libitum.
- Acclimation period: Males: Seven days before commencement of treatment. Females: 21 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

TIME SCHEDULE
- Experimental start date (Pre-study chemistry): 16 February 2017
- Animal arrival: Females 5 April 2017; males: 19 April 2017
- Treatment commenced: 26 April 2017
- Necropsy: Males 5 June 2017; females 15 to 19 June 2017
- Experimental completion date (Pathology data locked): 17 August 2017

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Vehicle:

propylene glycol

Details on oral exposure:

Method of preparation: The required amount of test item was ground in a mortar using a pestle and mixed with vehicle to produce a smooth paste. The suspension was transferred to a measuring cylinder, vehicle was added to achieve the final volume, and the suspension was transferred to a beaker and 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) until required for use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Homogeneity and Stability of Dose Formulations: The homogeneity and stability of the test item in propylene glycol formulations were demonstrated at 1 mg/mL and 300 mg/mL for 1 day at ambient temperature (15 to 25 ºC) and refrigerated for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 9% of the initial time zero value and the coefficient of variation was less than 5%.
- Concentration of Dose Formulations: The mean concentrations of test item in test formulations analyzed during the study were within 9% of nominal, confirming the accuracy of formulation.

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after minimum of five weeks of treatment (animals were killed in Week 6).
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose selection was based upon a range-finding study (please see RSS section 7.5.1 Supporting study, Envigo, 2018 (range-finding)).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. 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.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Detailed observations were performed according to the following schedule:
F0 males: Week 1 - daily, Week 2 onwards - once each week
F0 females: Week 1 - daily, Week 2 - onwards - at least once each week
- Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation, one to two hours after completion of dosing, as late as possible in the working day.
Detailed physical examination and arena observations:
- 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. Any deviation from normal was recorded with respect to the nature and degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
- Sensory reactivity and grip strength: 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 at Day 7-9 of lactation for females.
- The following measurements, reflexes and responses were recorded: Approach response, pinna reflex, auditory startle reflex, tail pinch response, and grip strength.
- Motor activity: 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).

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

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes.
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
- F0 animals: Weekly, from the day that treatment commenced until mating detected. Food consumption was not recorded for males and females during mating (Week 3), but recommenced for males in Week 4. From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

HAEMATOLOGY: Yes.
- Peripheral blood: the five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group (at termination).
- Volume: 0.5mL
- Analysed for the following:
Hematocrit (Hct)
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)
Mean cell hemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
- 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. 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)
Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes.
- Blood chemistry: collected after overnight withdrawal of food from the five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group (at termination).
- Volume: 0.7mL
- Plasma analysed for the following:
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.
Thyroid Hormone Analysis: All surviving F0 adult males and females (no samples were obtained from the animal which failed to litter), (at termination).
- Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
- Conditions F0 animals: Following overnight deprivation of food
- Anesthetic: F0 animals: Isoflurane
- Blood sample site: F0 adults: Sublingual vein
- Anticoagulant: Plasma samples (for TSH): K2EDTA. Tubes used for collection of samples did not contain separator gel. Serum samples (for T4): None (Greiner Minicollect tubes with clotting activators)
- Blood volume: Adult animals: 2 x 0.5 mL
- Processing: Plasma samples: Samples were kept on wet ice prior to centrifugation at 2000g for ten minutes at 4°C. All available plasma/serum was transferred to appropriately labelled polypropylene “cryo” tubes using micropipettes and deep frozen (-60 to -90ºC).

Sacrifice and pathology:

Necropsy
All adult animals were subject to a detailed necropsy. Any abnormality in the appearance or size of any organ and tissue 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 produce a viable litter: Day 25 after mating; F0 females: Day 14 of lactation.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
- Testes: Initially in modified Davidson’s fluid.
- Eyes: In Davidson’s fluid.

Histology
- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned. For bilateral organs, sections of both organs were prepared. Full List: The five lowest numbered surviving males and first five lactating females with a surviving litter in Groups 1 and 4.
- 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.

Organ Weights
- For bilateral organs, left and right organs were weighed separately.

Light Microscopy
- The five lowest numbered surviving F0 males and first five lactating F0 females with a surviving litter in Groups 1 and 4 (scheduled kill).
- All remaining F0 animals; abnormalities only.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

F0 Responses
- Detailed Physical Examinations: There were no signs seen during the weekly detailed physical examinations or in association with the dosing procedure that were considered to be treatment-related.
- Sensory Reactivity and Grip Strength: Sensory reactivity observations and grip strength were similar for animals treated at 100, 300 or 1000 mg/kg/day, when compared with the Controls.
- Motor Activity: Unaffected by treatment with the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Overall bodyweight and bodyweight change for males during treatment were unaffected by treatment with the test item.
During the first week of treatment females receiving 1000 mg/kg/day had no mean weight gain, compared with the Controls. Two females in each of Groups 1, 2 and 3 lost weight slightly (-13 to -3 g) in Week 1, but six females receiving 1000 mg/kg/day lost weight slightly (-8 to -2 g) over this period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on the food consumption of males during treatment or of females prior to pairing, during gestation or during lactation.

Food efficiency:

not specified

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The haematological investigation undertaken in Week 6 for males and on Day 14 of lactation for females did not reveal any findings that were considered related to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma did not reveal any treatment related effects in the females but plasma phosphorus concentrations were slightly high in males receiving 330 or 1000 mg/kg/day and plasma chloride concentrations were high in males receiving 1000 mg/kg/day. In the absence of any effects seen on male kidney weights and no remarkable histopathological findings reported in the male kidneys these small variations in plasma phosphorus and chloride were considered non-adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The evaluation of organ weights of males after 5 weeks of treatment and of females on Day 14 of lactation revealed the following:
Marginally lower body weight adjusted kidney weights in all test item treated female groups, but not in a dose-dependent fashion and therefore are considered as incidental.(absolute and body weight adjusted).
Marginally lower body weight adjusted brain weights in all test item treated male groups, but not in a dose-dependent fashion and therefore are considered as incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The macroscopic examination performed after at least 5 weeks of treatment revealed no test item related findings.
The incidence and distribution of findings were considered to be unrelated to treatment.
- incidental Findings: Pale kidneys were seen in several females across all groups and this finding correlated with the mineralisation seen in the kidneys at microscopic examination. Dark areas in the glandular mucosa of the stomach were also seen in a few females across all groups and this finding correlated with submucosal oedema/haemorrhage/necrosis seen in the glandular stomach at microscopic examination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No findings related to treatment were observed.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone analysis:
- There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Not applicable.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, oral administration of the test item to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was well-tolerated in the adult animals with no treatment related adverse effect observed. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg/day, the highest dose tested.

Executive summary:

The purpose of this study was to assess the potential systemic toxicity in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of the test item, by oral gavage administration for at least five weeks. This study can therefore be used to address the repeated dose toxicity endpoint.

 

Three groups of ten male and ten female rats received the test item at doses of 100, 330 or 1000 mg/kg/day in propylene glycol 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, propylene glycol, at the same volume dose as the treated groups.

 

During the study the following observations were made for the adult animals: 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 (T4), organ weight and macroscopic pathology and histopathology investigations were undertaken.

 

No animals died prematurely. Clinical condition, behavior in the arena, sensory reactivity, grip strength and motor activity were unaffected by treatment. There were no signs seen in association with dosing.

 

The body weight performance of the males was unaffected by treatment with the test item.  In Week 1 of treatment (prior to pairing) females receiving 1000 mg/kg/day had no mean weight gain when compared with the Controls. No effect on body weight was observed during the gestation period but during the lactation phase of the study females receiving 330 or 1000 mg/kg/day gained less weight when compared with the controls, but not in a dose-dependent fashion. These variations in body weights were transient, not considered adverse.

 

Food consumption was unaffected by treatment.

 

Haematological investigations did not reveal any findings that could be attributed to treatment.

The biochemical examination of the blood plasma revealed slightly high plasma chloride and phosphorus concentrations in males receiving 1000 mg/kg/day with phosphorus concentrations also marginally high in males receiving 330 mg/kg/day.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in the Day 13 offspring.

Slightly lower mean body weight adjusted kidney weights were seen in all test item treated female groups and slightly lower mean body weight adjusted brain weights were seen in all test item treated male groups, but these organ weight variations were not dose-dependent, had no correlated histopathological changes and were considered of no toxicological importance.

 

The macroscopic and microscopic examination of adult males and females did not reveal any findings related to treatment.

 

In conclusion, oral administration of the test item, to parental Sprague Dawley (Crl:CD(SD)) rats at dose levels of 100, 330 or 1000 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 14 of lactation in females was well-tolerated in the adult animals with no treatment related adverse effect observed.