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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:
27 June 2017 to ****
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

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
Limit test:
no

Test material

Constituent 1
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)
EC Number:
615-708-0
Cas Number:
72030-25-2
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)
Constituent 2
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-
Cas Number:
153128-45-1
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-
Constituent 3
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-
Constituent 4
Reference substance name:
Unspecified 2-ethylhexyl thiophosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl thiophosphate esters
Constituent 5
Reference substance name:
Unspecified 2-ethylhexyl phosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl phosphate esters
Constituent 6
Reference substance name:
Unspecified 2-ethylhexyl dithiophosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl dithiophosphate esters
Test material form:
liquid
Specific details on test material used for the study:
Purity: Assumed 100%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Labs; Raleigh, NC
- Age at first dose: 12 to 13 weeks
- Weight at first dose: Males: 449.4 to 526.1g; females: 217.7 to 297.4g
- Housing: Polycarbonate cages suspended on stainless steel racks. Animals were individually housed prior to cohabitation and after confirmation of mating. During cohabitation, one male and one female from the same group were housed together. Dams and pups were group housed.
- Bedding: Certified Sani Chips® hardwood bedding
- Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum
- Water: Filtered water was provided ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature Range: 20 to 26°C
- Humidity Range: 30 to 70%
- Light Cycle: 12-hour light/12-hour dark
- Air Changes: Minimum of 10 air changes per hour

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
The formulations were stirred before and during dosing and were stored in a refrigerator (3 to 5°C) until used for dosing.
Details on mating procedure:
After at least 14 days of vaginal lavage and dosing, one female from each group was cohabited with one male from corresponding group (1:1). The females were tested daily by vaginal lavage beginning on the day following cohabitation. The animals were separated when mating was confirmed or after 14 days. Day 0 of pregnancy (gestational day [GD] 0) was defined as the day on which mating evidence was confirmed (vaginal plug or presence of sperm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analysis of the dose formulations was performed, the test item was prepared in peanut oil at concentrations ranging from 25 to 500 mg/mL and analysed via a validated UV/VIS method, with detection at 332 nm.
The homogeneity and stability of the test item formulations in peanut oil was confirmed for at least ten days when stored at room temperature and refrigerated and protected from light.
Duration of treatment / exposure:
At least 28 days; 34 days for F0 males; 56 to 57 days for F0 females that were non-parturient/without evidence of mating; post natal day 13 for F0 dams and F1 pups.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
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, Smithers Avanza, 2018 (range-finding)).

Examinations

Parental animals: Observations and examinations:
Animal Observations/Measurements of F0 Males
- Physical Examinations:
Study Day 1 (prior to initiation of dosing)
Weekly thereafter
Prior to necropsy

- Cageside Observations:
≥ Twice daily

- Body Weights:
SD1 (prior to initiation of dosing)
Weekly thereafter
At time of unscheduled termination
Prior to necropsy (fasted)

- Food Consumption:
Weekly prior to pairing

- Functional Observation Battery (FOB):
Once on SD 31

Animal Observations/Measurements of F0 Females
- Physical Examinations:
Study Day 1 (prior to initiation of dosing)
Weekly thereafter until confirmation of mating
Unconfirmed females were returned to weekly exams following completion of cohabitation
Gestational Day 0, 7, 14, and 20
Postnatal Day 0, 4, 7, and 13
Prior to necropsy

- Cageside Observations:
≥ Twice daily

- Body Weights:
SD1 (prior to initiation of dosing)
Weekly thereafter until confirmation of mating
Gestational Day 0, 7, 14, and 20
Postnatal Day 0, 4, 7, and 13
At time of unscheduled termination
Prior to necropsy (fasted)

- Food Consumption:
Weekly prior to pairing
Gestational Day 0-7, 7-14, and 14-20
Postnatal Day 0-4, 4-7, and 7-12

- Functional Observation Battery (FOB):
Once on PND 11 or 12 (5 females/group)

Cageside observations included observation for mortality, moribundity, general health, and signs of toxicity.
Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns.
For the FOB, animals were transported to the testing room and acclimated to white noise for at least 10 minutes prior to testing.

Clinical Pathology
Samples were collected prior to each scheduled termination (scheduled animals only).
- Serum Clinical Chemistry:
Albumin
Alkaline Phosphatase
Alanine Aminotransferase
Aspartate Aminotransferase
Blood Urea Nitrogen
Calcium
Cholesterol
Creatine Kinase
Chloride
Creatinine
Gamma Glutamyltranspeptidase
Glucose
Potassium
Sodium
Phosphorus
Total Bile Acid
Total Bilirubin
Total Protein
Triglycerides

- Hematology:
> Complete Blood Count
White Blood Cells
Red Blood Cells
Hemoglobin
Hematocrit
Mean Corpuscular Volume
Mean Corpuscular Hemoglobin
Mean Corpuscular Hemoglobin Concentration
Mean Platelet Volume
Platelets
Red Cell Distribution Width
> Leukocyte Differential Count
Absolute Neutrophils
Absolute Lymphocytes
Absolute Monocytes
Absolute Eosinophils
Absolute Basophils
> Reticulocyte Count
Absolute Reticulocytes

- Hormone Analysis:
> Blood Collection from F0 Generation
On the day of necropsy, blood was collected from animals via cardiac puncture under 70% CO2/ 30% O2 inhalant anesthesia, at least 0.6 mL was collected into serum separator tubes and animals were fasted for sample collection.
Oestrous cyclicity (parental animals):
The stage of estrus was determined in each female for at least 14 days prior to dosing, 14 days prior to cohabitation, during cohabitation until evidence of mating or 14 days of cohabitation, whichever occurred first, and prior to necropsy by vaginal lavage.
Sperm parameters (parental animals):
The testis was stained with periodic acid-Schiff stain (PAS-H) and examined microscopically with special emphasis on stages of spermatogenesis and interstitial testicular cell structure.
Litter observations:
>Litter size and observations
The number (alive and dead), and the sex, weight, and clinical observations of individual pups, performed on PND 0, 4, 7 and 13
>Litter Culling
Culling to a total of 10 pups, preferably 5 males and 5 females. Individual pups identified by tattoo, performed on PND 4
> Anogenital Distance (AGD)
All pups, performed on PND 4
> Nipple Retention
Number of nipples/areolae were counted, performed on PND 13 (male pups)
Postmortem examinations (parental animals):
- Termination:
> Animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.
> Females without evidence of mating and non-parturient females necropsied (uterus and ovaries were examined) on Study Day 58 ± 2.
> On Study Day 35 for F0 males, Post Natal Day 13 for F1 pups and F0 females.
> Necropsy occurred on SD 35 for F0 males, PND 13 for F1 pups and F0 females, all surviving animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.

-Necropsy:
> Animals were necropsied as soon as possible after the time of death or discovery. Bone marrow smears were prepared (five F0 animals/sex/group), required organs were weighed, and protocol-specified tissues were collected and preserved. No organ weights were collected from animals found dead or euthanized prior to scheduled termination and no bone marrow smears were prepared from animals found dead.
> Gross necropsy of F0 animals included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. All reproductive organs (testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix; sex appropriate) were collected and weighed as soon as possible after dissection; paired organs were weighed together. For five selected animals/sex/group at scheduled necropsy, additional protocol-specified tissues were collected and weighed as soon as possible after dissection; paired organs were weighed together.
Tissues were preserved in 10% neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10% NBF. Tissues from animals found dead were preserved in 10% NBF only. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation.

- Histopathology:
> Preserved tissues from the five females and males selected in the control and high dose study groups were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically.
Postmortem examinations (offspring):
- Hormone Analysis:
> Blood Collection from F1 Generation (PND 4 and 13) for thyroid hormone analysis.
Blood was collected from culled pups on PND 4. Blood samples were pooled by litter. Culled pups were not fasted prior to collection.
On PND 13, blood was collected from at least two pups/litter at termination. Blood was not pooled by litter. Pups assigned to blood collection were discarded without necropsy.
Reproductive indices:
The following were calculated for each litter:

Male Mating Index (%) = No. of Males with Evidence of Mating / Total no. of Males Used for Mating x 100

Male Fertility Index (%) = No. of Males Siring a Litter / Total no. of Males Used for Mating x 100

Male Copulation Index (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating x 100

Female Mating Index (%) = No. of Females with Evidence of Mating / Total no. of Females Used for Mating x 100

Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / Total no. of Females Used for Mating x 100

Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating x 100
Offspring viability indices:
The following were calculated for each litter:

Mean Live Litter Size = Total Viable Pups on PND 0 / No. of Litters with Viable Pups on PND 0

Postnatal Survival between Birth and PND 0 or 4 (%) = Sum of Viable Pups per Litter on PND 0 or 4 / No. of Litters per Group x 100

Postnatal Survival = Sum of (Viable Pups per Litter at end of N) / No. of Litters per Group x 100
(Where N = PND 0 to 4 or PND 7 to 13)

Male Sex Ratio = No. of Male Pups at birth / Total No. of Pups Born

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Several animals were euthanized as moribund (one male at 700 mg/kg/day on SD 15 (gavage error); one female at 700 mg/kg/day on SD 3 (cause undetermined)) or found dead (one male at 100 mg/kg/day on SD 22 (cause undetermined); two females at 700 mg/kg/day, one each on PND 0 and PND 1(complications with parturition)). All other animals survived until the scheduled termination.

Observations from cageside or physical examinations were considered incidental and not test substance related due to no associated dose response. Control males exhibited abrasions, alopecia, hunched posture and rough haircoat. Among males surviving to scheduled termination, alopecia, rough haircoat, loud respiration noises and a stained fur were observed. Observations in the male euthanized due to gavage error were labored respiration, gasping, hunched posture, rough haircoat, salivation, and urine staining.

Among females surviving to scheduled termination, alopecia, abrasions, and abnormal respiration (loud or wheezing) were observed. One female at 700 mg/kg/day, observed with stained fur and continuous vocalization on SD 3, was euthanized in a moribund condition. The two Group 4 females that were euthanized in moribund condition on PND 0 or 1 were observed with severe ataxia, cold to the touch, hunched, languid, red discharge from the vagina, stained fur, rough haircoat, and pale. Other findings during lactation included low incidence of alopecia, abrasion, and swelling of the right inguinal region.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male that received 100 mg/kg/day was found dead on SD 22 and at 700 mg/kg was euthanized in a moribund condition on SD 15. The cause of death for another animal was determined to be a gavage error.

Two females that received 300 mg/kg/day were found dead during the study on GD 19 and three females were euthanized in a moribund condition on SD 3 and PND 1. The females euthanized on PND 0 and 1 were euthanized due to complications with parturition and it could not be determined if the deaths were related to the test item. All other animals survived until the scheduled termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No significant differences were noted in mean body weights, however mean body weights of Group 4 males (700 mg/kg) were consistently lower than that of control animals from SD 8 to 29 (-23.5% to -39.9%).

Mean body weights and body weight changes of females were comparable across groups throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test substance had no effect on food consumption.
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):
Males (Study Day 35)
Test substance-related changes consisted of minimal decreases (22%) in reticulocyte at 700 mg/kg/day. No correlating changes in the bone marrow or body weight were observed.
Decreases in measures of erythroid mass (red blood cell count, hemoglobin concentration and hematocrit percentage) occurred at doses ≥300 mg/kg/day, the cause for these decreases was uncertain and given the small magnitude of the change and lack of distinct relationship to dose were considered equivocal and could not be attributed to the test substance.

Females (Post Natal Day 13)
Test substance-related changes consisted of minimal decreases in reticulocyte at 700 mg/kg/day. No correlating changes in the bone marrow or body weight were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related changes consisted of minimal increases in alanine aminotransferase (ALti) activity in animals administered 300 or 700 mg/kg/day and in gamma glutamyl transpeptidase (GGT) activities in animals administered 700 mg/kg/day.

Females (Post Natal Day 13)
Test substance-related changes consisted of minimal increases in GGT activities in animals administered 700 mg/kg/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Treatment with the test item had no effect on the functional observation battery.

The only abnormal results was obtained for one animal, at 700 mg/kg/day, which had low activity and low posture during open field observations. No abnormal observations were noted for females. Quantitative measures (grip strength, hindlimb splay, grooms, fecal boli, urine pools, and rears) were not significantly different across groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic changes were observed in the thyroid at doses ≥ 100 mg/kg/day in males and at doses ≥ 300 mg/kg/day in females, in the liver in both sexes at doses ≥ 300 mg/kg/day and the stomach in males at doses ≥ 300 mg/kg/day and females at a dose level of 700 mg/kg/day.

In the stomach, hyperplasia of the mucosal epithelium with erosion or ulceration was observed in animals administered 700 mg/kg/day. One male administered 300 mg/kg/day had test item-related changes in the nonglandular stomach characterized by multifocal mild hyperplasia of the mucosa.

Another male given 300 mg/kg/day had minimal hyperkeratosis in hyperplastic areas and minimal neutrophilic inflammation in the keratin layers. These changes may have been a systemic effect of the test item, or secondary to direct irritation of the mucosa.

Increased absolute and relative liver weights correlated with these findings at doses ≥ 300 mg/kg/day in males, and at a dose level of 700 mg/kg/day in females. Notably, two females administered 300 mg/kg/day had minimal hepatocellular hypertrophy that did not correlate with increased group liver weight parameters; however, these two individuals had the highest liver weights in the group.

Correlating changes in clinical chemistry consisted of minimal increases in ALTi activity in males administered ≥ 300 mg/kg/day and in GGT activities in both sexes administered 700 mg/kg/day.

Thyroid follicular cell hypertrophy noted in males at doses ≥ 100 mg/kg/day and females administered ≥ 300 mg/kg/day. This change was considered most likely secondary to changes in the liver.

Of uncertain relationship to test item administration was minimal alveolar hemorrhage in the lungs of two males and one female administered 700 mg/kg/day; this finding may be seen as a common background change and was therefore considered unlikely to be related to the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Hormone Levels
Treatment with the test substance had no effect on T4 levels of males; no significant differences were noted.

Coagulation
No test substance-related changes in coagulation parameters occurred.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment with the test item had no effect on vaginal cytology. All females were cycling and regular prior to dosing.
Irregular cycling in Group 4 animals was primarily due to prolonged estrus, although mating success was not impacted; this was not considered test item related due to no associated dose response or a corresponding statistically significant change in cycle numbers and length.
The mean number of cycles during the 14-day cytology period ranged from 2.17 (Group 4) to 2.67 (Group 2). Mean cycle length ranged from 3.95 days (Group 1) to 4.34 days (Group 4).
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Description (incidence and severity):
Treatment with the test substance did not impact pregnancy rate or affect mating performance. Mating success ranged from 66.67% (Group 1) to 100% (Group 2; 100 mg/kg). The percent of dams that littered were 75% (Group 1), 100% (Group 2), 83.33% (Group 3; 300 mg/kg), and 100% (Group 4; 700 mg/kg). Mean gestation periods were comparable, ranging from 21.2 days to 22.3 days.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
700 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 performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
not specified

Results: F1 generation

Effect levels (F1)

Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test substance the test item did induce repeat dose toxicity, although reproductive toxicity was not affected in male and female rats. The repeat dose no adverse effect level (NOAEL) was considered to be 100 mg/kg/day due to adverse body weight changes, organ weight changes and microscopic findings of hepatocellular hypertrophy. The 700 mg/kg/day dose was the NOAEL for both the females and males for reproductive toxicity.
Executive summary:

The purpose of this study was to determine the potential toxicity of the test item when administered daily for at least 28 days via oral gavage to male and female Sprague Dawley rats and to determine potential reproductive and developmental toxicity.

 

Ninety six (48/sex) Sprague Dawley rats were randomly assigned to four groups (12 animals/sex). Animals were administered control substance (peanut oil) or the test item at 0, 100, 300, or 700 mg/kg once daily via oral gavage for at least 28 days. Animals were subjected to a full gross necropsy on Study Day (SD) 35 (F0 males), or Postnatal Day (PND) 13 (parturient F0 females and pups). Females that did not litter were subjected to a full gross necropsy on SD 57.

 

Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, vaginal cytology, clinical pathology (clinical chemistry, hematology, and coagulation), thyroid hormone (T4) analysis (males and PND 13 pups only), gross pathology findings, absolute and relative organ weights, and histopathology findings.

 

Treatment with the test item at doses ≥ 300 mg/kg had an effect on the liver. Test-substance related hepatocellular hypertrophy correlated with increased liver weight parameters and increases in hepatobiliary enzyme (GGT and/or ALTi) activities. Thyroid follicular cell hypertrophy in males at doses ≥ 100 mg/kg/day and females administered ≥ 300 mg/kg/day was considered most likely secondary to changes in the liver. Additionally, test substance-related changes in the nonglandular stomach consisted of mucosal hyperplasia with erosion or ulceration was observed in animals administered 700 mg/kg/day, mild mucosal hyperplasia in one animal administered 300 mg/kg/day and minimal hyperkeratosis and minimal neutrophilic inflammation in another animal administered 300 mg/kg/day. These changes may have been a systemic effect of the test substance or secondary to direct irritation of the mucosa.

 

There was no effect on mortality, physical examinations, cageside observations, body weights, food consumption, vaginal cytology, clinical pathology (hematology, coagulation, and urinalysis), thyroid hormone analysis (males and PND 13 pups), or gross pathology findings at all dose levels evaluated.

 

There was no reproductive toxicity observed at all dose levels, up to 700 mg/kg/day. In the males at doses up to 700 mg/kg/day, there were no differences in reproductive organ weights or reproductive parameters evaluated. In females at doses up to 700 mg/kg/day, there were no differences in estrus cycles, pregnancy, or conception rates.

 

In summary, the test substance the test item did induce repeat dose toxicity, although reproductive toxicity was not affected in male and female rats. The repeat dose no adverse effect level (NOAEL) was considered to be 100 mg/kg/day due to adverse body weight changes, organ weight changes and microscopic findings of hepatocellular hypertrophy. The 700 mg/kg/day dose was the NOAEL for both the females and males for reproductive toxicity.

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