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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:
06 February 2017 - 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

Constituent 1
Chemical structure
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 exposure:
Test substance formulations and vehicle control were stirred continuously at room temperaturefor the duration of the dosing.
Details on mating procedure:
The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females with the following exception. The 2 additional females added to Group 4 on Study Day 8 were only dosed for 6 days prior to pairing.
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:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
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.
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

Parental animals: Observations and examinations:
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.
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each female for 14 days prior to randomization and continuing until evidence of copulation was observed.
Sperm parameters (parental animals):
sperm morphology and the testes were palpated at least once for all males.
Litter observations:
Each litter was examined daily for survival, and all deaths were recorded

Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13.

Pups were individually weighed on PND 1, 4, 7, 10, and 13. Mean pup weights were presented by sex for each litter and by dose group.
Postmortem examinations (parental animals):
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
Postmortem examinations (offspring):
On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Blood samples were collected for thyroid hormone analysis immediately prior to euthanasiafrom 1 pup/sex/litter; gross necropsies were conducted and the thyroids (with parathyroids, if present) were placed in 10% neutral-buffered formalin for possible histopathological examination.
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.
Reproductive indices:
All females were allowed to deliver naturally and rear their young to PND 13. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia.
Offspring viability indices:
Litter parameters were defined as follows:
Mean Live Litter Size = Total No. of Viable Pups on PND 0/No. of Litters with Viable Pups PND 0

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

Postnatal Survival for All Other Intervals (% Per Litter) = Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)*/No. of Litters Per Group x 100

Where N = PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–10, 10–13, and 4 (post-selection)–13
* = Pups that were euthanized due to death of the dam were excluded from pup viability calculations.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
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
Food consumption and compound intake (if feeding study):
no effects 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
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat. (total fraction)
Sex:
male
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
mortality

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Three (3), 2(2), 1(1), and 2(1) pups (litters) in the vehicle control, 15, 50, and 150/100 mg/kg/day groups, respectively, were found dead. In addition, 12 pups from 1 litter were euthanized due to the death of dam. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects 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
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

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.