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Administrative data

Endpoint:
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:
other: OECD 422 Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat
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
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.
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.
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).
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

Maternal examinations:
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.
Ovaries and uterine content:
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.
Fetal examinations:
>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)
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: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, 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:
no mortality observed
Description (incidence):
Treatment with the test substance did not affect body weights for females. Mean body weights and body weight changes of females were comparable across groups throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Treatment with the test substance did not affect body weights for females. Mean body weights and body weight changes of females were comparable across groups throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
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):
Females (Post Natal Day 13)
Test substance-related changes consisted of minimal decreases in reticulocyte at a dose level of 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):
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, dosed at 700 mg/kg, which had low activity and low posture during open field observations. No abnormal observations were noted for females. Means of 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
Description (incidence and severity):
Females (Post Natal Day 13)
Test substance-related changes in females were limited to increases in absolute and relative liver weights at a dose level of 700 mg/kg/day. Increases were only statistically significant in absolute liver weights and liver weight relative to body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related macroscopic observations occurred.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic changes were observed in the thyroid at doses ≥ 300 mg/kg/day in females, in the liver at doses ≥ 300 mg/kg/day, and the stomach at a dose level of 700 mg/kg/day.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Coagulation
No test substance-related changes in coagulation parameters occurred.

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
not examined
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
Treatment with test item had no effect on litter sex or size. No significant difference in litter size was noted and the mean male pup ratio was comparable across groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
700 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
dead fetuses
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios

Maternal abnormalities

Key result
Abnormalities:
not specified

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Treatment with the test item had no effect on body weights of pups. Mean pup body weight increased over the course of the study. No statistically significant differences were observed between Group 1 and the other treatment groups on any post-natal day. No trend was noted in males, females or combined sexes.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Treatment with test substance had no effect on litter sex or size.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no gross abnormalities identified in any pup.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Treatment with the test substance had no effect on ano-genital distance.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
700 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test item did induce repeat dose toxicity, although developmental 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 the development of the offspring.
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 non-glandular 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, cage-side 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.

 

The test substance did not induce developmental toxicity at all dose levels evaluated. The litter size, pup weight and observations on the morphology of the pups were performed from birth to PND 13 and there was no indication that the test substance affected development.

 

In summary, the test item did induce repeat dose toxicity, although developmental 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 the development of the offspring.

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