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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
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. 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.
- 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
- 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
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Dosing was restricted to the F0 generation
- 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.
Details on mating procedure:
- Pairing commenced: After a minimum of two weeks of treatment.
- Male/female ratio: 1:1 from within the same treatment groups.
- Duration of pairing: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
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
Details on study schedule:
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.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
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, 2017 (range-finding)).

Examinations

Parental animals: Observations and examinations:
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.

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 which commenced within 30 minutes of sampling. Serum samples: Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation at 2000g for ten minutes at 4°C and deep frozen (-60 to -90ºC).
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.
Sperm parameters (parental animals):
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:
- 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 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.
Postmortem examinations (parental animals):
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.

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.
Postmortem examinations (offspring):
SACRIFICE
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled Kill: Day 13 of age.

GROSS NECROPSY and HISTOPATHOLOGY
- Performed on the five lowest numbered surviving males and first five lactating females with a surviving litter in Groups 1 and 4.
- Hematology, Peripheral Blood, Blood Chemistry: Performed at termination on the five lowest numbered surviving males and the first five lactating females with a surviving litter iin each dose group.
- Thyroid Hormone Analysis:
> Blood samples were collected as follows:
- Day 4 of age: F1 offspring, two females per litter
- Day 13 of age: F1 offspring, two males and two females per litter (where possible).
- All surviving F0 adult males and females (except the female which failed to litter) at termination.
Reproductive indices:
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Group mean values were calculated from individual litter values.
Offspring viability indices:
The following were calculated for each litter:

Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 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) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
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, when 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):
not examined
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 examined
Water consumption and compound intake (if drinking water study):
not examined
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.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects 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 or in offspring on Day 13 of age. There was therefore no requirement to measure T4 in the samples obtained from offspring on Day 4 of age or from the adult females and none of the TSH (thyroid stimulating hormone) samples required analysis.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Unaffected by treatment.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
Unaffected by treatment.
Reproductive performance:
no effects observed
Description (incidence and severity):
Unaffected by treatment.

Effect levels (P0)

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:
clinical signs
mortality
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance

Results: F1 generation

Effect levels (F1)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day.
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.

 

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, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis (T4), 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 recorded. Thyroid hormone analysis (T4) was performed for the Day 13 offspring. Nipple counts were performed on male offspring on Day 13 of age.

 

No animals died prematurely. Clinical condition was 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 mean weight in 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.

 

Estrous cycles, pre-coital interval, fertility, mating performance, gestation length and index were unaffected by treatment. There was no effect of treatment on the number of implantations or litter size.

 

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.

 

Reproductive performance, fertility and offspring survival were unaffected by parental treatment.  In the context of this study, the test item showed no evidence of being an endocrine disruptor. The no-observed-adverse-effect-level (NOAEL) for systemic toxicity and also for reproductive/developmental toxicity was considered to be 1000 mg/kg/day, the limit dose tested.