Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

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:

08 February 2017 to 29 March 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

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

EC Number:

270-220-1

EC Name:

Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

Cas Number:

68413-48-9

Molecular formula:

C28H55O6PS2

IUPAC Name:

1,4-dibutyl 2-({bis[(2-ethylhexyl)oxy](sulfanylidene)-λ⁵-phosphanyl}sulfanyl)butanedioate

Test material form:

liquid

Specific details on test material used for the study:

CAS RN 68413-48-9
Physical Description: Pale yellow, clear liquid
Purity: 84%

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 51 days old
- Weight at study initiation: Males: 293 to 415g; females: 210 to 286g
- Housing: 2–3 rats/cage by sex in solid-bottom cages with bedding material
- Diet: certified feed (PMI Nutrition International, LLC Certified Rodent LabDiet® 5002)
- Water: Reverse osmosis-purified (on-site) drinking water
- Fasting: All males and females (including those not selected for clinical pathology evaluation) were fasted prior to clinical pathology blood collection when food, but not water, was withheld
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): minimum of 10
- Photoperiod: 12-hour light/12-hour dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared in corn oil approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2ºC to 8ºC), protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations prepared at nominal test substance concentrations of 20, 60, and 200 mg/mL were analyzed using a validated high performance liquid chromatography (HPLC) method, using ultraviolet (UV) absorbance detection at a wavelength of 260 nm.
The results met the protocol-specified acceptance criteria for concentration acceptability for uniform solution formulations. No test substance was detected in the analyzed vehicle.

Duration of treatment / exposure:

Males were dosed on study days 0-27.
Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49-53 doses.

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.:

300 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

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels of 100, 300 and 1000 mg/kg bw/day were selected based on the results of a previous 14-day range-finding study. The test substance was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 10 males and 10 females. A concurrent vehicle control group of 10 rats/sex received the vehicle on the same regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test substance/vehicle administration. Males received 14 daily doses prior to mating and were dosed throughout the mating period until 1 day prior to necropsy for a total of 28 doses. Females received 14 daily doses prior to mating and were dosed through Lactation Day 13 for a total of 49-53 doses.

Examinations

Observations and examinations performed and frequency:

Clinical Observations and Survival
All animals were observed twice daily for moribundity and mortality. Clinical observations were recorded daily and detailed physical examinations were conducted weekly. Observations for signs of toxicity were recorded approximately 1.5 hours after dose administration. Functional observational battery (FOB) and motor activity data were recorded for 5 males/group during the last week of dosing (Study Day 27) and for 5 females/group on Lactation Day 13. Social groups were observed at appropriate intervals for findings that could not be attributed to a single animal.

Body Weights
Individual body weights were recorded weekly throughout the study for males, and weekly until evidence of copulation was observed for females. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, 13, and 14.

Food Consumption
Food consumption was measured on a per cage basis on the corresponding weekly body weight days until pairing, but was not recorded during the breeding period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, and 13.

Clinical Pathology
Blood samples for clinical pathology evaluations were collected from 5 animals/sex/group at the scheduled necropsies. The animals were fasted overnight prior to blood collection. Blood for serum chemistry and hematology was collected via the jugular vein for males and via the retro-orbital sinus following isoflurane anesthesia for females. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing K2EDTA (hematology), sodium citrate (coagulation), or no anticoagulant (serum chemistry).

The following parameters were evaluated:

> Hematology and Coagulation:
Total leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (Platelet)
Prothrombin time (PT)
Activated partial thromboplastin time (APTT)
Reticulocyte count
Percent (RETIC)
Absolute (RETIC Absolute)
Differential leukocyte count -
Percent and absolute
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Red cell distribution width (RDW)
Hemoglobin distribution width (HDW)
Platelet estimate
Red cell morphology (RBC Morphology)

> Serum Chemistry:
Albumin
Total protein
Globulin [by calculation]
Albumin/globulin ratio (A/G Ratio) [by calculation]
Total bilirubin (Total BILI)
Urea nitrogen
Creatinine
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma glutamyltransferase (GGT)
Glucose
Total cholesterol (Cholesterol)
Calcium
Chloride
Phosphorus
Potassium
Sodium
Sorbitol dehydrogenase (SDH)
Triglycerides (Triglyceride)
Bile Acids
Appearance (Includes degree of hemolysis, lipemia, and icterus)

Sacrifice and pathology:

Macroscopic Examination
A complete necropsy was conducted on all F0 parental animals at the scheduled termination. Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia for males and on Lactation Day 13 for females. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

Organ Weights
The following organs were weighed from all F0 animals at the scheduled necropsies:
Adrenal glands
Brain
Epididymides (paired organs were weighed separately)
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Prostate gland
Seminal vesicles (with coagulating gland and fluid)
Spleen
Testes (paired organs were weighed separately)
Thymus gland
Thyroids with parathyroids

Histology and Microscopic Examinations
After fixation, specified tissues were trimmed, processed into paraffin blocks, sectioned, mounted on glass microscope slides, and stained with hematoxylin and eosin. PAS staining was used for the testes and epididymides. Microscopic examination was performed on all tissues from 5 animals/sex in the vehicle control and 1000 mg/kg/day groups at the scheduled necropsies. Gross lesions were examined from all animals in all groups.

Other examinations:

Thyroid Hormone Analysis
Blood (target 1 mL/sample) was collected as described previously in serum chemistry into tubes without anticoagulant and allowed to clot at room temperature for at least 30 minutes. Serum was isolated in a refrigerated centrifuge and stored frozen (approximately -65ºC to -85ºC).
The following thyroid hormone parameters were evaluated for F0 males: Thyroxine (T4)
The samples from all F0 females (PND 4) were not analyzed.

Statistics:

Analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the vehicle control group by sex. Parental body weights, body weight changes, and food consumption, absolute and relative organ weights, clinical pathology values, thyroid hormone values, and FOB data values were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the vehicle control group. FOB parameters that yield scalar or descriptive data in the test substance-treated groups were compared to the vehicle control group using Fisher’s Exact test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical findings were noted at daily examinations following dose administration at any dosage level. Clinical findings, including hair loss, scabbing, and/or clear, red, or yellow material on various body surfaces, occurred infrequently, similarly in the vehicle control group, and/or in a manner that was not dose-related. No relationship to the test substance was evident.

Mortality:

no mortality observed

Description (incidence):

All F0 animals survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Males: Mean F0 body weights and body weight gains in the 100, 300, and 1000 mg/kg/day group males were unaffected by test substance administration throughout the study.
- Females: Mean F0 body weights and body weight gains in the 100, 300, and 1000 mg/kg/day group females were unaffected by test substance administration during the pre-mating period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean F0 food consumption in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration throughout the study (males) and during the pre-mating period (females).

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):

There were no test substance-related effects on F0 hematology and coagulation. Significant (p < 0.05 or p < 0.01) differences from the vehicle control group were higher mean percentage of basophils in the 100 mg/kg/day group males, lower mean percentage of monocytes in the 300 mg/kg/day group females, and shorter mean prothrombin time in the 100 mg/kg/day group females. No dose-response relationship was evident. Therefore, these findings were considered incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related effects on F0 serum chemistry parameters. Changes in serum chemistry consisted of lower mean cholesterol levels in the 1000 mg/kg/day group F0 males, higher mean albumin and total protein levels in the 1000 mg/kg/day group F0 females and higher mean A/G ratios in the 100 and 1000 mg/kg/day group F0 females. With the exception of the mean total protein level in the 1000 mg/kg/day group females, the differences from the vehicle control group were significant (p < 0.05). However, these changes did not correlate with other signs of toxicity or corresponding microscopic findings, and the magnitude of the changes were not toxicologically meaningful; therefore, the changes in serum chemistry were not considered test substance-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage observations, handling observations, open field observations, sensory observations, neuromuscular and physiological observations and motor activity were all unaffected by test substance administration.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in organ weights. However, some significant differences were observed when the vehicle control and test substance-treated groups were compared. The left testis to body weight ratio was higher in the 1000 mg/kg/day group males when compared to the vehicle control group. The mean absolute testis weight and mean testis weight relative to brain weight was not different than the vehicle control group. Therefore, the higher mean left testis weight relative to body weight was considered spurious. The mean kidney weight relative to final body weight was higher in the 1000 mg/kg/day group females when compared to the vehicle control group. The mean absolute kidney weight and mean kidney weight relative to brain weight was not different than the vehicle control group, and the higher mean kidney weight relative to body was considered spurious. Additional statistically significant differences were observed when the test substance-treated groups were compared to the vehicle control group; however, these findings lacked a clear dose-response relationship and were not considered test substance-related. Seminal fluid was lost prior to collection of the seminal vesicle/coagulating gland weights for 1 male each in the 100, 300, and 1000 mg/kg/day groups. Therefore, the weights of the seminal vesicle/coagulating glands of these 3 males were not included in the mean organ weight calculations, which did not affect the outcome of the study because the seminal vesicle/coagulating gland weights from the remaining 9 males each in the 100, 300, and 1000 mg/kg/day groups were sufficient for data interpretation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related internal findings were observed at any dosage level in 1 female in the vehicle control group that failed to deliver or males and females at the scheduled necropsy. Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related. The mean numbers of unaccounted-for sites and implantation sites in the 100, 300, and 1000 mg/kg/day groups were comparable to the vehicle control group values.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related histologic changes. Necrosis and inflammation of the arteries/arterioles (with thrombosis) in the submucosa of the cecum was observed in one 1000 mg/kg/day group female. This change was characterized by multifocal fibrinoid necrosis of the vessel wall with transmural infiltrates comprised of predominantly neutrophils and lymphocytes. In 1 vessel, a thrombus occludes the lumen. These changes were considered consistent with spontaneous necrotizing vasculitis (polyartertitis) that is occasionally observed in rodents and was not considered a test substance-related change. Minimal mononuclear infiltrates and minimal mineralization were observed in the 1000 mg/kg/day group female rats. These are well described background changes in the rat and were not considered test substance-related. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis (F0 Males)
There were no test substance-related effects on serum T4 levels in the F0 males at any dosage level. Differences from the vehicle control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

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:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats.

Executive summary:

The potential toxic effects of the test substance at dosage levels of 100, 300 and 1000 mg/kg bw/day were investigated in an OECD 422 study, performed under GLP conditions. The test substance or vehicle control (corn oil) was administered to rats via gavage once daily to 4 groups of Crl:CD(SD) rats, each group consisting of 10 males and 10 females.

 

All F0 animals survived to the scheduled necropsy. No test substance-related clinical findings were noted at the daily examinations or approximately 1.5 hours following dose administration at any dosage level. No test substance-related effects on mean F0 male and female body weights, body weight gains, or food consumption (including during gestation and lactation in females) were noted at any dosage level. No test substance-related effects on F0 neurobehavioral parameters (FOB and motor activity) or reproductive performance (male and female mating and fertility, male copulation, and female conception indices, mean estrous cycle and gestation lengths, pre-coital intervals, and the process of parturition) were observed at 100, 300, or 1000 mg/kg/day.

 

There were no test substance-related effects on hematology, serum chemistry or thyroid hormone (T4) parameters in the F0 males and females at any dosage level. No test substance-related macroscopic or microscopic findings were noted at any dosage level.

 

Under the conditions of this screening study, no adverse effects were noted in F0 adults at any dosage level. Therefore, 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for systemic toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats.