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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June-July 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
420-990-1
EC Name:
-
Cas Number:
146421-65-0
Molecular formula:
C20H36N2O6
IUPAC Name:
4-(ethenyloxy)butyl N-[6-({[4-(ethenyloxy)butoxy]carbonyl}amino)hexyl]carbamate
Constituent 2
Chemical structure
Reference substance name:
4-(ethenyloxy)butyl N-{6-[({[6-({[6-({[4-(ethenyloxy)butoxy]carbonyl}amino)hexyl]carbamoyl}oxy)hexyl]oxy}carbonyl)amino]hexyl}carbamate
Cas Number:
1516571-16-6
Molecular formula:
C34H62N4O10
IUPAC Name:
4-(ethenyloxy)butyl N-{6-[({[6-({[6-({[4-(ethenyloxy)butoxy]carbonyl}amino)hexyl]carbamoyl}oxy)hexyl]oxy}carbonyl)amino]hexyl}carbamate
Test material form:
solid: flakes
Details on test material:
- Name of test material (as cited in study report): Uralac P 1910C/Uralac P 1920C
- Physical state: solid: white flakes

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight range at start of treatment was 312-316 g (males) or 197-202 g (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
Lactation: Pups were kept with the dam until termination in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage-enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June-July 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. To obtain visual homogeneity, formulations were heated to a maximum of 92.7°C for a maximum of 59 minutes. Formulations were allowed to cool down to below 40°C before dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.

Storage conditions of formulations: at ambient temperature under nitrogen

Justification for use and choice of vehicle: based on trial formulations performed at WIL Research Europe

Dose volume: 10 mL/kg bw. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase, according to a validated method (Project 502162). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Results: The concentrations analysed in the formulations of the treatment groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the control formulation. The formulations of the low and high dose were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature for at least 6 hours.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. Detection of mating was not confirmed for two animals, which did deliver live offspring. The mating date fo these animals was estimated at 21 days prior to actual delivery.
Duration of treatment / exposure:
Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One female from each group was not dosed during littering (day 1 of lactation).

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w.
Duration of test:
Males: 28 days
Females: 41-45 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on a 28-Day study from a similar compound, URALAC ZW 3307 P (NOTOX Project 235261). Dose levels of 50, 200 and 1000 mg/kg bw/d were tested. Females at 1000 mg/kg had slightly lower body weights than controls; this was the only finding noted and the NOAEL was set at 1000 mg/kg bw/d. This study is used as a read-across for URALAC P 1910C / URALAC P 1920C.

Parturition:
The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Identification of pups:
On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT
- Time schedule for examinations:Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

GROSS PATHOLOGY
Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
The number of former implantation sites and corpora lutea were recorded for all paired females.

- All females which failed to deliver or had total litter loss: According to test guidelines

HISTOPATHOLOGY
According to test guidelines: ovaries of females from control group and high dose and reproductive organs from females that failed to deliver; all gross lesions of all animals.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
SACRIFICE
Pups surviving to planned termination were killed by decapitation on day 5 of lactation.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
No.

The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.
- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS: Yes
If possible, defects or cause of death were evaluated.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. No statistical analysis was performed on histopathology findings.
Indices:
Reproductive indices; For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition

Offspring indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100
Historical control data:
None reported

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted. Salivation seen after dosing among all animals at 1000 mg/kg bw/d, several animals at 500 mg/kg bw/d and one male at 100 mg/kg bw/d during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred. One female at 500 mg/kg bw/d and one female at 100 mg/kg bw/d were sacrificed due to total litter loss. No cause for the loss of the litter could be established histopathologically.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect on body weight and body weight gain was observed.
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
No toxicologically relevant changes in food consumption were noted.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic findings.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were considered unaffected by treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
One female at 1000 mg/kg bw/d did not become pregnant. Since a cause of non-pregnancy could not be established histopathologically for males and females, other females of this dose group did become pregnant and the incidence of this occurrence remained within the normal control range of non-pregnancies, this was considered unrelated to treatment with the test substance.
The apparent slightly lower number of corpora lutea and implantation sites at 1000 mg/kg bw/d were attributed to lower counts for a female that only delivered two pups, and a female that had total litter loss). Low numbers of corpora lutea and/or implantation sites were also noted for one control female, one female at 100 mg/kg bw/d and one female at 500 mg/kg bw/d, none of which delivered live offspring. Since other pregnant females of the 1000 mg/kg bw/d group showed counts that were considered to be within the normal control range, this was considered not to be of toxicological relevance.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects up to and includinh highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered to have been unaffected by treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 5 pups of the control group, 2 pups at 100 mg/kg bw/d, 12 pups at 500 mg/kg (of which 11 were due to total litter loss from one female, which resulted in a statistically significantly higher postnatal loss and lower viability index at this dose) and 1 pup at 1000 mg/kg bw/d (due to total litter loss of one female) were found dead or missing during lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and, except for the total litter loss of female, remained within the range considered normal for pups of this age.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Incidental macroscopic findings among pups that were found dead included advanced stage of autolysis, absence of milk in the stomach and cannibalism. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Other effects:
no effects observed
Description (incidence and severity):
Incidental clinical symptoms among surviving pups consisted of scabbing on the ear or front leg, lean appearance, absence of milk in the stomach and a blue spot on the back or neck. Two pups of a litter at 500 mg/kg bw/d found dead showed autolysis or cannibalism at last litter check. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Details on embryotoxic / teratogenic effects:
There was no evidence of teratogenic effects based on the absence of relevant clinical signs and external macroscopic findings. The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were considered unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effecst observed up to and including the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In an oral OECD 421 screening study with rats, the maternal and developmental NOAEL was determined to be 1000 mg/kg bw/day, based on the absence of effects observed.
Executive summary:

In an oral OECD 421 screening study rats were given 0, 100, 500 or 1000 mg/kg bw/d of URALAC P 1910C/URALAC P 1920C formulated in propylene glycol for 7 days/week for 28 days (males) and 41 -45 days (females). No adverse effects were observed on mortality, clinical signs, body weight( gain) and food consumption, macroscopy and histopathology. Reproduction parameters consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition and maternal care showed no effects. Sex ratio and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were not affected.

Based on the absence of adverse effects, the NOAEL for maternal and developmental toxicity was determined to be at least 1000 mg/kg bw/day.

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