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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
In-life phase: 07 April 2018 to 27 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)

Test material

1
Chemical structure
Reference substance name:
p-tert-butylstyrene
EC Number:
217-126-9
EC Name:
p-tert-butylstyrene
Cas Number:
1746-23-2
Molecular formula:
C12H16
IUPAC Name:
p-tert-butylstyrene
Test material form:
liquid: volatile
Specific details on test material used for the study:
Lot/Batch no.: 111617TBSSV-2
Purity: 96.4%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Age at first dose: 8-9 weeks
Weight range at mating (GD 0): 180-210 g
Weight range at first dose: 197.9-245.9 g
Animals were acclimated to laboratory conditions for a minimum of 1 day prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information. Animals were individually housed.

Temperature: 20 to 26°C
Humidity: 30 to 70%
Light/Dark cycle:12-hour light/12-hour dark
Air changes: Minimum of 10 air changes per hour
Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum.
Water: Filtered water was provided ad libitum via an automatic watering system supplemented with water bottles as needed.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
The formulations were placed at room temperature prior to dosing. The animals were dosed daily on GD 5 to 20 (day of confirmation of mating = GD 0) via oral gavage at a volume of 2.0 mL/kg. Dose volumes were based on the most recent body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analysis of the dose formulations was performed as part of the method validation. Test substance formulations were solutions; therefore, homogeneity sampling and analysis was not required. Samples from dose formulations prepared for week 1 and last week were analyzed for concentration verification using a validated method.
Details on mating procedure:
Time-mated female Sprague Dawley rats were received by the test facility.
Duration of treatment / exposure:
The animals were dosed daily on GD 5 to 20 (day of confirmation of mating = GD 0).
Frequency of treatment:
Daily once
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were initially accepted into the randomization pool based upon GD 0 body weights provided by the vendor. The suitability of the randomized animals was confirmed by physical examinations upon arrival. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p ≤ 0.05) from the control mean. Following randomization each study animal was assigned a unique number and identified by an ear tag.

Examinations

Maternal examinations:
Cageside observations included observation for mortality, morbundity, general health, and signs of toxicity (only abnormal findings were recorded during the cageside observations). 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.
Ovaries and uterine content:
The abdominal cavity was opened, the uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions for each uterine horn, and the number of total implantations were recorded. The number of corpora lutea on each ovary was also recorded. The embryonic membrane of each fetus was gently removed. The fetuses were removed from the uterus and the placentae were grossly examined. Each implant was categorized as viable, nonviable, late resorption, or early resorption. Uteri from females that appeared to be nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. The foci, if detected, were considered early resorptions, and data from these females were not included in mean calculations and statistics. If no foci were seen, the female was considered to be non-pregnant and data from these females were also not included in mean calculations or statistics.
Fetal examinations:
All live fetuses were individually weighed, identified, sexed, and examined for external malformations and variations. Following completion of the external examination of all fetuses in the litter, each fetus was euthanized. Approximately one-half of the fetuses in each litter were examined viscerally by fresh tissue dissection. The sex of the fetus was recorded. The heads were examined by Wilson’s sectioning. The remaining fetuses had the internal sex recorded and then were eviscerated, preserved, stained with Alizarin Red S and Alcian Blue, and examined for skeletal abnormalities. Fetal findings were classified as malformations or developmental variations. All fetal malformations were photographed, digitally saved and archived with the study file.
Intact fetuses from dams that died within 24 h of scheduled euthanasia were evaluated to the extent possible for external malformations and variations and sexed. All other intact fetuses from females not surviving to scheduled euthanasia were examined externally to the fullest possible extent and discarded.
Statistics:
See under "any other information on materials and methods incl. tables".

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Significant reduction in gravid uetrine weight which corresponded to reduced body weights.
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
mortality

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: general systemic toxicity

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Reduction in number of live offspring:
no effects observed
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
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Reduced body weight and reduction in body weight gain
Description (incidence and severity):
observed at all dose levels

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the NOAEL for maternal systemic toxicity was determined to be 20 mg/kg bw/day. No NOAEL for fetal developmental effects could be determined due to effects at all doses and therefore, the LOAEL for fetal developmental toxicity was considered to be 20 mg/kg bw/day.
Executive summary:

A prenatal developmental toxicity study was conducted in Sprague Dawley rats according to OECD Guideline 414, in compliance with GLP. The test substance was administered to groups of 25 time-mated Sprague Dawley female rats at 0, 20, 60 or 100 mg/kg bw/day through oral gavage from Gestation Day 5 to 20 (GD 5 to GD 20). Dams were subjected to a full gross necropsy and uterine and fetal examinations on GD 21. Parameters evaluated during the study included mortality, physical examinations, cageside observations, body weights, body weight changes, food consumption, gross pathology examinations, uterine data, and fetal examination data (external, visceral, skeletal, and head exams). Dose concentration analysis of samples from formulations prepared for Week 1 and the last week of dosing confirmed that the test substance was properly formulated. Administration of the test substance resulted in maternal mortality at 100 mg/kg bw/day due to body weight loss, decreased food consumption and associated clinical signs of rough haircoat and thin appearance. At 60 mg/kg bw/day, the adverse effects included reduced food consumption and body weight in the dams. There were no effects on pregnancy and uterine parameters including the number of live fetuses as a percentage of post-implantation sites. There was an increase in the incidence of fetal defects (malformations and variations) in all the treated groups. These skeletal variations included incomplete ossification and unossification of the sternebrae and cervical and thoracic centrum and were attributed to the increase in the incidence of unossified or incomplete ossification. These skeletal variations were considered secondary to the reduction in fetal body weight and associated with delayed ossification. Due to their reversible nature, these variations were considered non-adverse, as the offspring would continue to grow and the ossification process would continue until complete with no expected structural alterations. Under the study conditions, the NOAEL for maternal systemic toxicity was determined to be 20 mg/kg bw/day. No NOAEL for fetal developmental effects could be determined due to effects at all doses and therefore, the LOAEL for fetal developmental toxicity was considered to be 20 mg/kg bw/day (Murphy, 2019c).