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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recognised OECD test guideline and according to Good Laboratory Practice.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Benzyl, C7-C9 linear and branched alkyl esters, 1,2 benzene
dicarboxylic acid
- Sponsor designation: Santicizer 261A
- CAS No: 68515-40-2
- EC No: 271-082-5
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: 9E3009
- Vehicle: Certified Purina 5002 Meal
- Storage condition of test material: Tightly closed container in a cool, well-ventilated area

Test animals

Species:
rat
Strain:
other: Sprague Dawley Crl:CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: ~ 8 to 10 weeks upon arrival
- Weight at study initiation: ~ 225-250 g on GD 3 (day of randomization into groups at RTI)
- Number and sex: 100 timed-mated females; 25 timed-mated (presumed pregnant) females per group and 4 groups
- Mating: Mated at the vendor over 3 days to produce 24, 48, and 28 timed-mated females
- Identification: Microchipped (Bio Medic Data Systems, Seaford, DE) upon arrival at RTI
- Housing: Solid-bottom, polycarbonate caging; Acclimation and gestation: singly
- Enrichment: Nestlets
- Diet (e.g. ad libitum): Purina Certified Rodent Chow No. 5002 (Purina Mills, Inc., Richmond, IN)
Analysis : Provided by supplier Storage: 60-70°F in ARF
Expiration: 6 months after milling date
- Water (e.g. ad libitum): Tap water available ad libitum via an automatic water delivery system (Edstrom Industries, Inc., Waterford, WI)
Source: City of Durham, NC
Analysis reports: Annually by the City of Durham;
Twice yearly by independent laboratory analyses
- Husbandry: All animals used in compliance with the NRC Guide for the Care and Use of Laboratory Animals (2011).
- Bedding: Sani-Chips cage litter (P.J. Murphy Forest Products, Montville, NJ)
- Acclimatation: At least 3 ± 1 days under RTI Animal Research Facility (ARF) conditions. Release by RTI veterinarian for use on study at least 48 hours after arrival.
- Animal Welfare: Humane termination by CO asphyxiation if moribund or evidence of spontaneous abortion or premature delivery. Animals were not subjected to undue pain or distress.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72°F ± 3°F
- Humidity (%): 50 ± 20% RH
- Air changes (per hr): 10-15 per hour, except during scheduled maintenance
- Photoperiod (hrs dark / hrs light): 12-hour light:12-hour dark

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on analytical verification of doses or concentrations:
A prestudy chemistry was conducted to determine the dietary dose formulations of S-261A in feed.
The doses of 0, 250, 750, and 3750 ppm were accurate (99.2 – 107.2%), homogeneous, and stable for at least 42 days.
The dose formulations were therefore made once and used throughout this study with ad libitum exposure to the maternal animals from GD 6 through scheduled necropsy on GD 20.
Duration of treatment / exposure:
15-day
Frequency of treatment:
Dosed feed or vehicle was available ad libitum 7 days/week from GD 6, with adjustments of feed jars on GD 12 and 18.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Dose / conc.:
250 ppm
Dose / conc.:
750 ppm
Dose / conc.:
3 750 ppm
No. of animals per sex per dose:
Treatment groups: 3
Vehicle control group: 1
Number of animals per group: 25 timed-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
Females were assigned to treatment groups by stratified randomization, by body weight on GD 3, in order to provide uniform mean body weights across dose groups (±20%).
The initial weight range criterion of ±10% was changed to ±20% due to the variance in body weights observed on GD 3 (randomization time). These changes were considered to have no impact on this study.

Treatment groups: 3
Vehicle control group: 1
Number of animals per group: 25 timed-mated females

Dosing Regimen
On GD 6 through 20, S-261A in vehicle or vehicle alone was administered in the feed.
The Study Director and the Sponsor selected the highest concentration (3750 ppm; ~250 mg/kg/day) as one which was expected to induce maternal toxicity or low levels of lethality (< 10%).
The mid dose (750 ppm; ~50 mg/kg/day) was selected based on the expectation of pup developmental and systemic toxicity.
The low dose (250 ppm; ~16.7 mg/kg/day) was anticipated to be a maternal and embryo-fetal NOAEL in this study.
Dosed feed or vehicle was available ad libitum 7 days/week from GD 6, with adjustments of feed jars on GD 12 and 18.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: time the clinical sign was observed
severity (if applicable)
duration (if applicable) of the sign.

Daily morbidity/mortality checks were done twice per day, at least 6 hours apart, beginning the day after receipt.

Maternal observations:
-GD 20 euthanasia by CO2 asphyxiation
-Final body weight (g)
-Organ weights: uterus, liver
-Ovarian corpora lutea counts
-Uterine implantation sites classified as live, dead, early, late, or full resorption
-Dead fetuses (died in utero) recorded as dead and discarded
- Identification and retention of any maternal gross lesions in 10% formalin

Histopathology
Maternal livers were fixed in 10% neutral buffered formalin for subsequent optional histopathology. The formalin-fixed maternal livers from the control (0 ppm) and high dose (3750 ppm) groups were embedded in paraffin and sectioned.
The liver sections were placed on glass slides, stained with hematoxylin and eosin, and coverslipped for histopathologic evaluation.

Ovaries and uterine content:
See Maternal examination box
Fetal examinations:
Euthanasia
Live fetuses were dissected from the uterus and immediately placed on a moist paper towel over a tray of ice. This procedure induces anesthesia and subsequent death by lowering the core body temperature below 25°C (Lumb and Jones, 1973; Blair, 1979).

Fetal Observations:
Fetal Weight and Sex: All live fetuses
External Exam: 100% live fetuses
Fresh Visceral Exam: 50% live fetuses (decapitated)
Fixed Head Examination (Bouin’s): 50% (same fetuses which received visceral examination)
Alizarin Red S/Alcian Blue Skeletal
Staining: 100% (50% intact, 50% decapitated)
Skeletal Evaluation: 50% (intact fetuses)
Storage for Archiving Heads: 70% ethanol
Statistics:
All data were collected on the validated RTI Instem Provantis™ System and software except for fetal external, visceral, and skeletal findings.
For all statistical tests, p< 0.05 was used as the criterion for significance.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Statistically significantly increased absolute and relative liver weights was observed in Group 4 (3750 ppm) maternal females. No evidence of hepatocellular hypertrophy (increase in cell size), or hyperplasia (increase in cell number) were observed in the high dose animals.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
750 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
3 750 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Maternal Results

 

Pregnancy: There were 24, 25, 25, and 25 actual timed-pregnant females at 0, 250, 750, and 3750 ppm. 1 in the 0 ppm group was not pregnant.

 

Maternal surviving and abortion: All of the females survived to scheduled termination on GD 20 (no unscheduled deaths) and none of the pregnant females aborted or had a totally resorbed litter.

 

Mean maternal body weights: females were all statistically and biologically equivalent across all groups on GD 3 when they were assigned to groups and on GD 6, 9, 12, 15, 18, and 20 during their gestational exposure period. 

 

Mean maternal body weight gains: Statistically and biologically equivalent on GD 3-6 (prior to start of exposures), 6-9, 9-12, 12-15, 15-18, 18-20, 6-20 (exposure period), and 3-20 (on study).

 

Mean maternal feed consumption in g/animal/day: was equivalent across all groups for all intervals (corresponding to the body weight gain intervals) except for statistically significant increases at 750 ppm (p<0.01) and 3750 ppm (p<0.001) only for GD 12-15. 

 

Mean feed consumption values in g/day:±SEM for that interval were 21.01±0.73 at 0 ppm, 22.40±0.41 at 250 ppm, 23.46±0.48 at 750 ppm, and 24.11±0.58 at 3750 ppm. 

 

Mean maternal feed consumption in g/kg body weight/day for the same gestational intervals was statistically and biologically equivalent across all groups for GD 3-6, 6-9, 9-12, 15-18, and 18-20. However, the maternal feed consumption values were again statistically significantly increased on GD 12-15 for 750 ppm (p<0.01) and 3750 ppm (p<0.001). In addition, maternal feed consumption (g/kg/day) was significantly increased on GD 6-20 at 750 ppm and at 3750 ppm (both at -<0.05) and on GD 3-20 at 750 and 3750 ppm (both at p<0.05).

 

Maternal S-261 intake in mg/kg body weight/day: anticipated increases across groups (with highest values on GD 6-9, lowest values on GD 18-20) for all intervals calculated. For the 3-day intervals, the ranges were 0 mg/kg/day at 0 ppm, 16.775-20.672 mg/kg/day at 250 ppm, 50.539-65.489 mg/kg/day at 750 ppm, and 242.410-314.702 mg/kg/day at 3750 ppm. 

For overall intake on GD 6-20, the mean values were 0.0±0.0, 18.766±0.214, 57.572±0.796, and 287.770±4.016 mg/kg/day at 0, 250, 750, and 3750 ppm, respectively.

 

Maternal clinical observations: 1 female each at 0 and 3750 ppm with scabs, no clinical observations at 250 ppm, and 1 female with alopecia and 1 female with a damaged tail at 750 ppm.

 

Maternal gravid uterine weight, terminal body weight, terminal (GD 20) body weight minus gravid uterine weight, and body weight changes from GD 3-20: all statistically equivalent across all groups.

Maternal liver weights at scheduled necropsy, both absolute and relative to terminal body weights:

-       0, 250, and 750 ppm: equivalent across all groups. 

-       3750 ppm: significantly increased absolute maternal liver weight (by 7.01%) at p<0.01; significantly increased of relative maternal liver weight (by 7.60%) at p<0.001.

 

Histopathology was performed on the high dose and control maternal livers. No maternal hepatic macroscopic (gross) lesions in any livers in any of the groups. No evidence of hepatocellular hypertrophy or hyperplasia in the high dose livers.

 

Note of the pathologist: typically there is no hepatic hypertrophy or hyperplasia until the livers from the treated group(s) are at least 20% heavier than the control livers. In the present study the statistically significant and biologically relevant increases in maternal liver weights were 7.01% (absolute weight) and 7.60% (relative weight) higher than the control livers at the top dose, 3750 ppm. 

It is not known whether the maternal liver weights at the high dose would have increased above the ~7% increases observed, with a longer dosing period.

 

Developmental Toxicity Results

All maternal females had one or more live fetuses at scheduled necropsy on GD 20. 

Number of ovarian corpora lutea, number of uterine implantations per female: equivalent across all groups. 

 

Preimplantation loss/female (number of ovarian corpora lutea minus number of uterine implantation sites), percent preimplantation loss/female: low and equivalent across all groups.

 

No full litter resorptions in any female at any dose was observed. One dead fetus in 1 female at 3750 ppm was recorded.

Incidence of early resorptions and postimplantation loss: low and equivalent across all groups.

 

Mean numbers of live fetuses/litter: equivalent across all groups (11.7, 11.7, 11.4, and 11.4 at 0, 250, 750, and 3750 ppm, respectively;

Percentage of implantations which were live fetuses at term: high and equivalent across all groups (95.56, 97.66, 94.35, and 96.94% at 0, 250, 750, and 3750 ppm, respectively).

 

Alive foetuses/group: 0 ppm: 280; 250 ppm: 292; 750 ppm: 284; 3750 ppm: 285 live

Fetal body weights, expressed as total litter weight, fetal weight per litter (both sexes), and male and female fetal body weight/litter: all equivalent across all groups. 

Percent of male fetuses, relative to the total number of foetuses: equivalent across all groups (51.07, 50.34, 52.46, and 49.47% at 0, 250, 750, and 3750 ppm, respectively.

 

External foetuses malformations and variations.

 

Approximately 50% of each litter was examined for malformations.

No differences across groups for the litter or fetal incidences of external malformations or variations or for incidences of fetal skeletal malformations were observed.

 

There were also no differences across groups for litter or fetal incidences of total malformations or variations, regardless of type (e.g., external, visceral, or skeletal), with 35.7, 38.0, 39.8, and 38.6% of the fetusesin totoexhibiting variations per group and 1.5, 0.7, 3.5, and 2.1% of the fetusesin totoexhibiting malformations per group.

 

There was 1 fetus in 1 litter at 750 ppm with bilateral misshapen limbs, 1 fetus in 1 litter at 3750 ppm with bilateral hydroureter, a different fetus in 1 litter at 3750 ppm with left hydroureter, and 1 fetus in 1 litter each with right hydroureter at 250 and 750 ppm.  

 

Fetal skeletal malformations included: misshapen interparietal bone (3 fetuses in litter at 750 ppm), agenesis of a rib (1 fetus in 1 litter each at 0, 250, 750, and 3750 ppm), discontinuous rib (1 fetus in 1 litter each at 0 and 3750 ppm), fused rib (2 fetuses in 1 litter at 0 ppm), rib cartilage branched (1 fetus in 1 litter at 0 ppm), rib cartilage fused (2 fetuses in 1 litter at 0 ppm), thoracic centrum, bipartite ossification, bipartite cartilage (1 fetus in 1 litter at 750 ppm), and thoracic centrum, dumbbell ossification, and bipartite cartilage (1 fetus in 1 litter at 3750 ppm). 

The remaining fetal findings were classified as variations “not classified” (noted only) and observed in fetuses across all groups.

Applicant's summary and conclusion

Conclusions:
A developmental toxicity study was conducted on the test material S-261A, which was administrated to rats at 0, 250, 750, and 3750 ppm. Only at 3750 ppm a maternal hepatic toxicity in the form of increased absolute and relative body weight was observed. Necropsy proved no evidence of hepatocellular hypertrophy or hyperplasia. In addition, no adverse findings were observed in the prenatal offspring at any dose.
Executive summary:

The aim of the study was to investigate the effects ofin uteroexposure to Santicizer 261A (S-261A) on the organogenesis and prenatal development of the CD ®  rat. The test material was administrated to maternal animals for 15-day via the diet during the embryonic and fetal periods (gestational days [GD] 6 through 20). 

The only maternal findings of note were the statistically significantly increased absolute and relative liver weights observed in Group 4 (3750 ppm) maternal females. No evidence of hepatocellular hypertrophy (increase in cell size), or hyperplasia (increase in cell number) were observed in the high dose animals. 

No other treatment- or dose-related maternal findings were observed at any dose or any time during gestation or scheduled necropsy. No effects on maternal body weights, weight gains, feed consumption in g/day or g/kg/day, clinical observations, or pregnancy indices were recorded. No treatment- or dose-related developmental toxicity findings at any dose were observed including no effects on pre- or postimplantation loss, fetal numbers, sex ratio, body weights, or fetal external, visceral, or skeletal malformations or variations.