Dibutyl [(dipropoxyphosphinothioyl)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:

06 February 2017 to 03 April 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 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Cas Number:

68413-47-8

Molecular formula:

C18H35O6PS2

IUPAC Name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Test material form:

liquid

Specific details on test material used for the study:

- Description: Light yellow, clear liquid
- Storage: Room temperature (18 - 24°C), protected from light

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.
- Age at first dose: Approximately 10 weeks of age at the initiation of dose
administration.
- Weight at first dose: 326 g to 414 g and female body weights ranged from 218 g to 262 g on Study Day 0
- Housing: Clean, solid-bottom cages with bedding material (Bed O’Cobs®)
- Bedding: See above
- Feed: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 offered ad libitum
- Water: Filtered water was provided ad libitum
- Acclimation period: A minimum of 7 days

ENVIRONMENTAL CONDITIONS
- Temperature Range: 23°C ± 3°C
- Humidity Range: 50 ± 20%.
- 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:

corn oil

Details on oral exposure:

Test substance formulations and vehicle control were stirred continuously at room temperaturefor the duration of the dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of test substance in dosing formulations, including the vehicle control, were assessed on the first, middle, and last formulation prepared that included all dose groups.

Four 1.0 mL samples were collected from the middle strata for concentration assessments. Two samples from each strata were analyzed to assess the concentration of the test substance in the mixtures; the remaining samples were stored refrigerated (2°C to 8°C) as backup samples.

The acceptable result for a concentration assessment is a mean concentration within 100% ± 15% (85-115%) of the target concentration.

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.

Frequency of treatment:

Animals were dosed once daily for at least 14 days prior to mating.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Females were administered the test substance at a dosage level of 150 mg/kg/day from Study Days 0–12, inclusively. On Study Day 13, the dosage level for females was reduced to 100 mg/kg/day. The dosage level for the males remained at 150 mg/kg/day.

Doses / concentrations 5

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Females were administered the test substance at a dosage level of 150 mg/kg/day from Study Days 0–12, inclusively. On Study Day 13, the dosage level for females was reduced to 100 mg/kg/day.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The dosage levels were determined from results of a previous 14-day range-finding study in which the test substance

Examinations

Observations and examinations performed and frequency:

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at specified intervals.

FOB and motor activity data were recorded for 5 males/group during the last week of dose administration (Study Day 27) and for 5 females/group on Lactation Day 13.

F1 clinical observations, body weights, and sexes were recorded at specified intervals and anogenital distance was recorded on Postnatal Day (PND) 1.

To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (2/litter). All F1 male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F1 pups were euthanized on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were retained from 1 pup/sex/litter.

Clinical pathology evaluations (hematology, coagulation, and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. Blood samples for thyroid hormone analysis were collected from F0 males at necropsy and from F0 females on the day of the last dose (Lactation Day 13); only male samples were analyzed. F0 males were euthanized following completion of the mating period and F0 females were euthanized on Lactation Day 14 for females that delivered and Postmating Day 25 for females that failed to deliver. Complete necropsies were conducted on all F0 animals, and selected organs were weighed.

Selected tissues were examined microscopically from 5 F0 animals/sex in the vehicle control and high-dose groups.

Sacrifice and pathology:

A complete necropsy was conducted on all F0 parental animals found dead, euthanized in extremis, or at the scheduled termination.

All F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.


Females that delivered were euthanized on Lactation Day 14; blood samples were collected for thyroid hormone analysis on the day prior to euthanasia

Other examinations:

Organs were weighed from all F0 animals at the scheduled necropsies. Paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported. When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data.

Statistics:

All statistical tests were performed using WTDMS™. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals(N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Body weights, body weight changes, and absolute and relative organ weights were subjected to a parametric one-way ANOVA3 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 control group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Adverse clinical observations at the daily examinations were noted for these two females including ataxia, tremors, hypoactivity, and/or thin body. A female in the 150/100 mg/kg/day group was euthanized in extremis on Lactation Day 1 following adverse clinical observations at the daily examinations including hypoactivity, tremors, ataxia, clonic convulsions, thin body, and red material around nose at the daily examinations.

Test substance-related increased incidences of red and/or clear material around the mouth and nose were noted for males in the 150 mg/kg/day group at approximately 1.5 hours following dose administration primarily during the last weeks of dose administration (Study Days 13–27). These observations were generally resolved by the next scheduled observations and were not considered adverse. Other clinical observations noted in the test substance-treated groups, including hair loss or scabbing on various body surfaces, occurred infrequently, at similar frequencies in the vehicle control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, treatment-related

Description (incidence):

Seven females in the 150/100 mg/kg/day group were found dead or euthanized in extremis during the course of the study, with 4 out of 7 attributed to the test material.

A female in the 150/100 mg/kg/day group was found dead on Gestation Day 22.This death was attributed to an intubation error and was not test substance-related.

A female in the 150/100 mg/kg/day group was inadvertently removed from the animal room, and therefore this accidental death was not attributed to test substance administration.

In the 50 mg/kg/day group, a female was euthanized in extremis on Study Day 9 with findings indicative of an intubation error (epicardial, myocardial, and pleura inflammation), and therefore this moribundity was not test substance-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males:
Mean body weights and body weight gains in the 15, 50, and 150 mg/kg/day group males were unaffected by test substance administration throughout the study

Females:
Mean body weight gains in the 150/100 mg/kg/day group females were slightly lower (not statistically significant) than the vehicle control group during the premating period (Study Days 0–13). This difference was primarily due to a significantly (p < 0.05) lower mean body weight gain during Study Days 8–13. The decrements in mean body weight gain in the 150/100 mg/kg/day group were considered test substance-related, but were not of sufficient magnitude to affect mean body weights, and therefore were not considered adverse.

Mean body weights and body weight gains in the 15 and 50 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 food consumption, evaluated as g/animal/day, in the 15, 50, and 150 mg/kg/day group males was similar to that in the vehicle control group throughout the study. No statistically significant differences were observed.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher heart weights (absolute and relative to brain and body weights) were noted in the 150/100 mg/kg/day group females. There were no histologic alterations to correlate with this finding.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At the scheduled necropsies, review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. Notable findings included multiple cysts in the
kidneys, as well as bile duct dilation with peribiliary fibrosis in 2 animals. These findings were consistent with inherited polycystic kidney disease and not considered to be test substance-related. 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:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

2

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these results, the NOAELs for systemic toxicity were considered to be 150 mg/kg/day for males, the highest dose tested, and 50 mg/kg/day for females.

Executive summary:

The objectives of the study were to investigate the potential toxic effects of the test material when administered to rats for a minimum of 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behavior, and conception through day 13 of postnatal life.

Under the conditions of this screening study, no test substance-related effects were observed on F0 male and female fertility and mating indices, F0 male copulation index, F0 female conception index, gestation length, parturition, or reproductive organs at any dosage level. Therefore, dosage levels of 150 mg/kg/day for males and 100 mg/kg/day for females, the highest doses tested, were considered to be the no-observed-adverse-effect levels (NOAEL) for reproductive toxicity. Test substance-related mortality, moribundity, and adverse clinical observations (hypoactivity, tremors, ataxia, clonic convulsions, and thin body) were noted for females at 150 mg/kg/day, resulting in the reduction of the dosage level to 100 mg/kg/day on Study Day 13.

Following the reduction in dosage level for females, a single female in the 150/100 mg/kg/day group was euthanized in extremis. There were no adverse test substance-related clinical observations for males at 150 mg/kg/day or effects on body weights and food consumption for males and females at any dosage level. Test substance-related higher heart weights (absolute and relative to brain and body weights) were noted in the 150/100 mg/kg/day group F0 females. There were no adverse effects on clinical pathology parameters, thyroid hormones, or macroscopic/microscopic alterations in F0 males and females at any dosage level.

Based on these results, the NOAELs for systemic toxicity were considered to be 150 mg/kg/day for males, the highest dose tested, and 50 mg/kg/day for females. The NOAEL for neonatal toxicity was 100 mg/kg/day, the highest dose tested, based on the absence of effects on F1 offspring at all dosage levels.