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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
Study period:
September 23, 2014 to October 19, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
DeviationThe following unplanned deviation occurred as the result of an unintended deviation from the protocol.On one occasion, the Study Director was not provided gestation body weights and detailed clinical observations for 13 animals prior to selecting them for use on study. These data were reviewed after the randomization was performed, and all animals were considered acceptable for use on study.In the opinion of the Study Director, this deviation did not affect the quality or integrity of the study.
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear, yellow liquid
Details on test material:
Date Received: June 16, 2014
Supplier: Chemtura Fords, New Jersey
Amount Received: 2,083.3332 mL (2 containers)
Label Identification: Hatcol 3346
Batch Number: 2013059135
Physical Characteristics: Clear, yellow liquid
Retest Date: May 30, 2017
Storage: Room temperature, protected from light
TMC Number: 1405A8

Test animals

Species:
rat
Strain:
other: CD® [Crl:CD®(SD)]
Details on test animals and environmental conditions:
Animal Acquisition and AcclimationA total of 125 female CD® [Crl:CD®(SD)] rats (approximately 10.5 weeks of age) were received from Charles River Laboratories, Raleigh, North Carolina, on September 15, 2014.During the 1-week acclimation period, the animals were observed twice daily with respect to general health and any signs of disease. All animals were given a detailed clinical examination and body weights were recorded prior to selection. The Study Director reviewed body weight and detailed observation data for all animals and gave final approval for assignment to study. The findings from the pretest clinical examinations and body weights recorded during the acclimation period are not reported but are maintained in the study file.A total of 60 male CD® [Crl:CD®(SD)] rats (approximately 10.5 weeks of age) were received from Charles River Laboratories, Raleigh, North Carolina, on September 15, 2014. Body weight values for the males were recorded at receipt. The male animals were used for mating purposes only and any data recorded are not reported but maintained in the study file.Following the mating period, all males were transferred to a stock colony.Randomization, Assignment to Study, and MaintenanceFollowing the 1-week acclimation period, one male was housed with one female in the cage of the male. Positive evidence of copulation was established by daily inspection for a copulatory plug in situ or vaginal lavage for sperm. The day on which positive evidence of copulation was observed was considered GD 0. After mating, each female was returned to an individual cage. A total of 100 mated females were assigned to the control and treatment groups by consecutive placement in a block design (first mated female assigned to Group 1, second mated female assigned to Group 2, etc.). The order in which the animals were assigned corresponded to the day on which positive evidence of mating was observed.Extra female animals obtained, but not placed on study, were euthanized via carbon dioxide inhalation. Euthanasia was confirmed via exsanguination of the abdominal vena cava, and the carcasses were discarded.Each animal was assigned an animal number to be used in the Provantis™ data collection system and was implanted with a microchip bearing a unique identification number. The individual animal number, implant number, and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.The animals were individually housed in solid bottom cages with nonaromatic bedding in an environmentally controlled room except during pairing. Animal enrichment was provided according to SOP. Fluorescent lighting was provided for approximately 12 hours per day.Temperature and humidity were continuously monitored, recorded, and maintained to the maximum extent possible within the ranges of 68 to 79 °F and 30 to 70%, respectively. The actual temperature and humidity findings are not reported but are maintained in the study file.Block Lab Diet® (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was available ad libitum. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. The results of food and water analyses are retained in the Archives. The Study Director is not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of the study. Therefore, no analyses other than those stated above were conducted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Fresh vehicle, corn oil, was dispensed for use on study weekly under yellow lighting and was stored at room temperature and protected from light.The test article, Hatcol® 3346, was used as received from the Sponsor, and no adjustment was made for purity. Formulations of the test article were prepared weekly under yellow lighting at nominal concentrations of 20, 60, and 200 mg/mL, and were stored at room temperature and protected from light.The vehicle and test article were administered once a day from GD 0 to 19 at approximately the same time each day (±2 hours from the GD 0 dose) via oral gavage. The dose levels for the treated groups were 100, 300, and 1000 mg/kg/day at a dose volume of 5 mL/kg. The control group received the vehicle in the same manner as the treated groups. The dosing formulations were stirred throughout administration. Individual doses were based on the most recent body weights.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dosing FormulationsDosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see table under Any other information) were collected using a positive displacement pipette, while stirring, and placed into amber glass scintillation vials.AnalysesSamples were stored at room temperature until analyzed. All analytical work was conducted by MPI Research, Inc., using an analytical method developed and validated under MPI Research, Inc., Study Number 1038-049. No deviations from the analytical method occurred during the course of this study.ANALYTICAL SUMMARYObjective: To determine the homogeneity and concentration of Hatcol® 3346 in dosing formulations.Compliance: This nonclinical laboratory study phase was conducted in accordance with the United States Environmental Protection Agency (EPA), Toxic Substance Control Act (TSCA) Good Laboratory Practice (GLP) Standards, 40 Code of Federal Regulations (CFR) Part 792, and the Organization for Economic Cooperation and Development (OECD) Principles on GLP [as revised 1997; ENV/MC/CHEM(98).Analytical Method Number and Title: 1038-049: Determination of Hatcol® 3346 in Corn Oil Dose Formulations by HPLC/UVReference Standard: Hatcol® 3346Lot Number: 2013059135MPI Research Inventory ID: 1405A8 (SP5003085)Storage: Room temperature, protected from lightCorrection Factor: noneData Collection and Analysis Software: Empower™ 2: Build 2154 (Waters Technologies Corporation®); ExyLIMS Version 3.0 (MPI Research, Mattawan, Michigan)HPLC Conditions: Liquid chromatography system equipped with an Ascentis Express RP-Amide column, 4.6 x 100 mm, 2.7 μm particle size with a gradient flow of 0.1% phosphoric acid in water (mobile phase A) and 0.1% phosphoric acid in acetonitrile (mobile phase B) at a flow rate of 1.5 mL/minute.Analysis Description: Prior to analysis, duplicate samples were diluted with isopropyl alcohol (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 100. An aliquot of each sample was injected into the HPLC-UV system for analysis and evaluated at 225 nm.Regression Type: Linear, unweightedStudy Sample Receipts: Number of Samples: 72Dates of receipt by Analytical Department: September 22, 2014 to October 8, 2014Study Sample Storage Conditions: AmbientStorage Stability: All samples were analyzed within the established storage stability determined under MPI Research Study Number 1038-049(1).Analysis Period: September 23, 2014 to October 10, 2014Run Acceptance CriteriaSystem Suitability Test Standards: 1. Injection repeatability (peak area and retention time) ≤2% RSD (relative standard deviation)2. Resolution between the analyte peak and any adjacent peaks must be ≥1.53. Tailing Factor (T) ≤24. Theoretical Plates (N) ≥2000Calibration Standards: 1. Accuracy within ±5% of the nominal concentration2. Coefficient of determination (R2) ≥0.995 Performance Check Standards (Same preparation as the System Suitability Standard):1. Periodically injected so that no more than 10 samples are bracketed by performance check standards. Samples are considered valid if bracketed by passing performance check standard injections.2. Accuracy within ±5% of the nominal concentration; Blank Injections: ≤20% of the effective limit of quantitation (ELOQ)AssessmentsHomogeneity: 1. Average concentration within ±10% of the nominal concentration2. Precision ≤5% RSDConcentration: 1. Average concentration within ±10% of the nominal concentration2. Precision ≤5% RSD3. Vehicle (control) samples < ELOQResults and DiscussionConclusion: A total of 24 samples were analyzed for Hatcol® 3346 in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results.
Details on mating procedure:
Following the 1-week acclimation period, one male was housed with one female in the cage of the male. Positive evidence of copulation was established by daily inspection for a copulatory plug in situ or vaginal lavage for sperm. The day on which positive evidence of copulation was observed was considered GD 0. After mating, each female was returned to an individual cage.
Duration of treatment / exposure:
The vehicle and test article were administered once a day from GD 0 to 19 at approximately the same time each day (±2 hours from the GD 0 dose) via oral gavage.
Frequency of treatment:
Daily
Duration of test:
19 days
No. of animals per sex per dose:
25 mated females per dose group (100 in total)
Control animals:
yes, concurrent vehicle
Details on study design:
Justification of Test SystemThe current state of scientific knowledge and the applicable guidelines cited previously do not provide acceptable alternatives, in vitro or otherwise, to the use of live animals to accomplish the purpose of this study. “The development of knowledge necessary for the improvement of the health and well-being of humans as well as other animals requires in vivo experimentation with a wide variety of animal species.” “Whole animals are essential in research and testing because they best reflect the dynamic interactions between the various cells, tissues, and organs comprising the human body.”The rat is the usual rodent model used for evaluating the toxicity of various classes of chemicals and for which there is a large historical database. This laboratory has historical control data on the incidence of fetal malformations and variations and other fetal endpoints in this strain from this source. This strain is susceptible to known developmental toxicants.Justification for Route of AdministrationThe oral route is the probable route of exposure of this test article in humans.Justification of Dose LevelsThe dose level was selected by the Sponsor, or in consultation with the Sponsor, on the basis of available data from a pilot study in rats (MPI Research, Inc., Study Number 1038-044).Dose levels in this pilot study were 250, 500, 750, and 1000 mg/kg/day. No maternal toxicity was observed in this study, and no effects were observed on uterine implantation data, fetal sex ratios, fetal body weights, or fetal external examinations. Since the pilot study involved small group sizes and did not include further evaluations of the fetuses (i.e., no visceral or skeletal examinations), the high dose in the definitive study was selected as 1000 mg/kg/day and 100 and 300 mg/kg/day for the low and intermediate dose levels, respectively.AdministrationThe vehicle and test article were administered once a day from GD 0 to 19 at approximately the same time each day (±2 hours from the GD 0 dose) via oral gavage. The dose levels for the treated groups were 100, 300, and 1000 mg/kg/day at a dose volume of 5 mL/kg. The control group received the vehicle in the same manner as the treated groups. The dosing formulations were stirred throughout administration. Individual doses were based on the most recent body weights.

Examinations

Maternal examinations:
In-life ExaminationsCage side ObservationsAll animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily. On one occasion, a veterinary consultation was conducted prior to the study start. All treatments and observations were recorded. Detailed Clinical ObservationsDaily from GD 0 through 20 (approximately 60 to 90 minutes post dose on dosing days), each animal was removed from the cage and given a detailed clinical examination. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.Body Weights and Body Weight ChangesBody weights for all animals were measured and recorded on GD 0, 3, 6, 9, 12, 15, 18, and 20. Individual body weight change was calculated for the following GD intervals: 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-20, and 0-20. Adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to 20) were also calculated.Food ConsumptionFood consumption was measured and recorded on the corresponding body weight days and calculated for the same intervalsPostmortem Study EvaluationsMaternal NecropsyA complete necropsy was performed on all females under procedures approved by a veterinary pathologist. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy. The presence of lesions or other abnormal conditions in the dam were noted and described in the study records.
Ovaries and uterine content:
Maternal necropsies and subsequent fetal evaluations were conducted without knowledge of the treatment groups in order to minimize bias.On GD 20, each female was euthanized by carbon dioxide inhalation, followed by exsanguination of the abdominal vena cava and immediately subjected to a cesarean section. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened, and the uterus was exposed. 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 total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.Each implant was categorized according to the following criteria.Viable fetuses responded to touch. Nonviable fetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterized by recognizable fetal form, but undergoing autolysis. Early resorptions were characterized as implantation sites that had no recognizable fetal characteristics.The fetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each fetus was gently removed, and each fetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites4. If no foci were detected, the female was considered to be nonpregnant.
Fetal examinations:
Each fetus was individually weighed, sexed, tagged, and examined for external malformations and variations. Fetuses were then euthanized by intraperitoneal injection of euthanasia solution.Approximately one-half of the fetuses in each litter were placed in Bouin’s solution and the remaining fetuses were fixed in alcohol. All fetuses fixed in Bouin’s solution were examined for soft tissue defects using the Wilson razor-blade sectioning technique5. The fetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S, and cleared with glycerin by a method similar to that described by Dawson6 for subsequent skeletal examination. Fetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist. On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments are not reported, but are maintained in the study data. Mechanical artifacts (i.e., tail removed and discarded) occurred during examination and processing of the fetuses.
Statistics:
Detailed under any other information
Indices:
Fetal and litter incidences are reported, but only the litter incidences were statistically analyzed.
Historical control data:
The laboratory has historical control data on the incidence of fetal malformations and variations and other fetal endpoints in this strain from this source.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No effect of Hatcol® 3346 at the dose levels evaluated (100, 300, and 1000 mg/kg/day) was observed from the post dose detailed clinical examinations. Clinical findings observed among animals in the treated groups occurred at low incidence or with comparable frequency as controls and were considered unrelated to the test article.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled termination on GD 20.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effect of Hatcol® 3346 at the dose levels evaluated was observed on gestation body weights and body weight change. Mean body weights and body weight change in the treated groups throughout gestation were comparable to mean control values.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effect of Hatcol® 3346 at the dose levels evaluated was observed on gestation food consumption. In general, mean food consumption in the treated groups during gestation was comparable to mean control values. The one exception was a statistically significant increase in food consumption over GD 18 to 20 in the 300 mg/kg/day dose group relative to the mean control value (25.80 g/animal/day vs. 23.52 g/animal/day in controls). This was considered a spurious occurrence and unrelated to the test article. For all other intervals during gestation to include GD 0 to 20, mean food consumption in the 300 mg/kg/day dose group was comparable to mean control values.
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:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the maternal macroscopic examinations. The few findings observed among the treated animals occurred at low incidence or with similar frequency as controls and were considered unrelated to the test article.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No effect of Hatcol® 3346 at the dose levels evaluated was observed from corpora lutea and uterine implantation data. Uterine parameters to include mean number of implantation sites, viable fetuses, nonviable fetuses, litter size, resorption sites (early, late, and total), and pre and post-implantation loss indices in the treated groups were comparable to mean control values. Likewise, mean gravid uterine weights, adjusted GD 20 body weights, and adjusted body weight change GD 0 to 20 in the Hatcol® 3346-treated groups were comparable to mean control values.
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:no effectsDetails on maternal toxic effects:In-life ExaminationsMortalityAll animals survived to scheduled termination on GD 20.Detailed Clinical ObservationsNo effect of Hatcol® 3346 at the dose levels evaluated (100, 300, and 1000 mg/kg/day) was observed from the post dose detailed clinical examinations. Clinical findings observed among animals in the treated groups occurred at low incidence or with comparable frequency as controls and were considered unrelated to the test article.Body Weights and Body Weight ChangesNo effect of Hatcol® 3346 at the dose levels evaluated was observed on gestation body weights and body weight change. Mean body weights and body weight change in the treated groups throughout gestation were comparable to mean control values.Food ConsumptionNo effect of Hatcol® 3346 at the dose levels evaluated was observed on gestation food consumption. In general, mean food consumption in the treated groups during gestation was comparable to mean control values. The one exception was a statistically significant increase in food consumption over GD 18 to 20 in the 300 mg/kg/day dose group relative to the mean control value (25.80 g/animal/day vs. 23.52 g/animal/day in controls). This was considered a spurious occurrence and unrelated to the test article. For all other intervals during gestation to include GD 0 to 20, mean food consumption in the 300 mg/kg/day dose group was comparable to mean control values.Postmortem Study EvaluationsUterine and Ovarian ExaminationsPregnancy Indices were 92%, 100%, 88%, and 92% in the control, 100, 300, and 1000 mg/kg/day dose groups providing 23, 25, 22, and 23 litters, respectively with fetuses for evaluation on GD 20.No effect of Hatcol® 3346 at the dose levels evaluated was observed from corpora lutea and uterine implantation data. Uterine parameters to include mean number of implantation sites, viable fetuses, nonviable fetuses, litter size, resorption sites (early, late, and total), and pre and post-implantation loss indices in the treated groups were comparable to mean control values. Likewise, mean gravid uterine weights, adjusted GD 20 body weights, and adjusted body weight change GD 0 to 20 in the Hatcol® 3346-treated groups were comparable to mean control values.Maternal Macroscopic ObservationsNo effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the maternal macroscopic examinations. The few findings observed among the treated animals occurred at low incidence or with similar frequency as controls and were considered unrelated to the test article.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No effect of Hatcol® 3346 at the dose levels evaluated was observed from fetal body weights. Mean fetal body weights distinguished by sex and for the combined sexes, in the treated groups were comparable to mean control values.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No effect of Hatcol® 3346 at the dose levels evaluated was observed from fetal body weights. Mean fetal body weights distinguished by sex and for the combined sexes, in the treated groups were comparable to mean control values.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect of Hatcol® 3346 at the dose levels evaluated was observed from fetal sex ratios (% male fetuses/animal). Mean sex ratios in the treated groups ranged from 44.0% to 50.7% and were comparable to the 47.0% in the control group.
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):
No effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the fetal external examinations. The external malformations observed among fetuses in the 100 and 1000 mg/kg/day dose levels were dissimilar, occurred at low incidence (one fetus with tail and anus defects in the 100 mg/kg/day dose group and hind limb polydactyly in one fetus in the 1000 mg/kg/day dose group) or with similar frequency as in the controls (i.e., a single occurrence of omphalocele observed in one fetus in the 100 mg/kg/day fetus and also observed in one control fetus). The occurrence of these malformations was considered spontaneous in nature and unrelated to the test article. No external malformations were observed in fetuses from the 300 mg/kg/day dose group. No external developmental variations were observed among fetuses in the treated groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the fetal skeletal examinations. The few skeletal malformations observed among fetuses in the treated groups (i.e., malformations of the sacral and caudal vertebrae in the one fetus in the 100 mg/kg/day dose group with a tail defect, dissimilar sternebrae defects in two fetuses in the 300 mg/kg/day dose group, and additional phalanges in the one fetus observed externally with polydactyly in the 1000 mg/kg/day group) were considered spontaneous in nature and unrelated to the test article. Skeletal developmental variations observed in the treated groups occurred at low incidence or with similar frequency as in controls and no effect of the test article was indicated from these data.
Visceral malformations:
no effects observed
Description (incidence and severity):
No effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the fetal visceral examinations. No visceral malformations were observed among fetuses from the treated groups. In the control group one fetus had a folded retina. Dilated ureter, a visceral developmental variation, was observed in a single fetus in each of the 100 and 300 mg/kg/day dose groups. This variation has been observed in recent historical control data for the laboratory and its low incidence in these groups was considered incidental and unrelated to the test article. Dilated ureter was not observed in fetuses from the 1000 mg/kg/day dose group.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:Fetal Sex RatioNo effect of Hatcol® 3346 at the dose levels evaluated was observed from fetal sex ratios (% male fetuses/animal). Mean sex ratios in the treated groups ranged from 44.0% to 50.7% and were comparable to the 47.0% in the control group.Fetal Body WeightsNo effect of Hatcol® 3346 at the dose levels evaluated was observed from fetal body weights. Mean fetal body weights distinguished by sex and for the combined sexes, in the treated groups were comparable to mean control values.External ExaminationsNo effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the fetal external examinations. The external malformations observed among fetuses in the 100 and 1000 mg/kg/day dose levels were dissimilar, occurred at low incidence (one fetus with tail and anus defects in the 100 mg/kg/day dose group and hind limb polydactyly in one fetus in the 1000 mg/kg/day dose group) or with similar frequency as in the controls (i.e., a single occurrence of omphalocele observed in one fetus in the 100 mg/kg/day fetus and also observed in one control fetus). The occurrence of these malformations was considered spontaneous in nature and unrelated to the test article. No external malformations were observed in fetuses from the 300 mg/kg/day dose group. No external developmental variations were observed among fetuses in the treated groups.Visceral ExaminationsNo effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the fetal visceral examinations. No visceral malformations were observed among fetuses from the treated groups. In the control group one fetus had a folded retina. Dilated ureter, a visceral developmental variation, was observed in a single fetus in each of the 100 and 300 mg/kg/day dose groups. This variation has been observed in recent historical control data for the laboratory and its low incidence in these groups was considered incidental and unrelated to the test article. Dilated ureter was not observed in fetuses from the 1000 mg/kg/day dose group.Skeletal ExaminationNo effect of treatment with Hatcol® 3346 at the dose levels evaluated was observed from the fetal skeletal examinations. The few skeletal malformations observed among fetuses in the treated groups (i.e., malformations of the sacral and caudal vertebrae in the one fetus in the 100 mg/kg/day dose group with a tail defect, dissimilar sternebrae defects in two fetuses in the 300 mg/kg/day dose group, and additional phalanges in the one fetus observed externally with polydactyly in the 1000 mg/kg/day group) were considered spontaneous in nature and unrelated to the test article. Skeletal developmental variations observed in the treated groups occurred at low incidence or with similar frequency as in controls and no effect of the test article was indicated from these data.

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects noted

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Any other information on results incl. tables

Homogeneity

Analysis of the low- and high-dose formulations (20.0 and 200 mg/mL, respectively) used for dosing the first week of study confirmed they were homogeneous as prepared, meeting the laboratory’s acceptance criteria of 100±10% of nominal, and percent relative standard deviation (RSD) ≤5. These data are summarized in the table below.

Homogeneity

Dose Level (mg/kg/day)

Nominal Concentration (mg/mL)

Average Calculated Concentrationa(mg/mL)

Average %Recoverya,b

%Relative Standard Deviation

100

1000

20.0

200

20.4193

204.1510

102.1

102.1

1.279

1.249

aRepresents an average of six samples from the mixing container (2 top, 2 middle and 2 bottom). Samples were collected while the formulation was stirring.

bAverage % recovery was calculated from the nominal concentration.

 

Concentration

Mean concentrations of formulations used for dosing in Weeks 1 and 3 of study ranged between 100.4 and 103.2% of nominal with percent RSDs ranging between 0.006 and 1.306, confirming that animals were receiving the appropriate dose levels when the formulation was administered at 5 mL/kg. No test article was detected in the control samples. These data are summarized in the table below.

Concentration

Dose Level (mg/kg/day)

Nominal Concentration (mg/mL)

Average Calculated Concentrationa(mg/mL)

Average %Recoverya,b

%Relative Standard Deviationa

0

100

300

1000

0.00

20.0

60.0

200

BLQ

20.3066 – 20.4360

60.2423 – 61.0501

202.2807 – 206.3500

NA

101.5 – 102.2

100.4 – 101.8

101.1 – 103.2

NA

0.464 – 1.279

0.006 – 1.306

1.000 – 1.264

aResults are the range of values determined during Weeks 1 and 3.

bAverage % recovery was calculated from the nominal concentration.

BLQ – Below the Limit of Quantification (<2.0000 mg/mL)

NA – Not Applicable

 

Summary of gestation Detailed Clinical Observations+

Days 0 to 20

Observation

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Number of Animals Observed

25

25

25

25

Animal Husbandry

               Nail missing, Forefoot/left

               Nail missing, Forefoot/right

 

0/0

0/0

 

17/1

17/1

 

0/0

0/0

 

0/0

0/0

Pelage/Skin

               Abrasion(s), Forefoot/right

               Hair sparse, Abdominal region

               Hair sparse, Cervical region

               Hair sparse, Face

               Hair sparse, Forefoot/left

               Hair sparse, Forefoot/right

               Hair sparse, Forelimb/left

               Hair sparse, Forelimb/right

               Hair sparse, Hind limb/left

               Hair sparse, Hind limb/right

               Hair sparse, Lumbar region

               Hair sparse, Tail

               Ventral surface

               Scabbed area, Cervical region

               Scabbed area, Cranial region

               Scabbed area, Face

               Scabbed area, Forelimb/left

               Scabbed area, Lumbar region

               Scabbed area, Thoracic region

 

3/1

10/2

10/1

0/0

11/1

25/2

29/5

29/5

8/1

1/1

0/0

0/0

0/0

0/0

3/1

0/0

0/0

12/3

0/0

 

0/0

0/0

0/0

16/1

34/3

18/2

8/1

0/0

0/0

0/0

0/0

0/0

0/0

48/4

0/0

12/1

0/0

0/0

4/1

 

0/0

0/0

13/3

0/0

10/2

10/2

31/4

38/5

11/1

0/0

7/2

0/0

11/1

18/2

0/0

0/0

0/0

0/0

6/1

 

0/0

4/1

12/1

0/0

32/5

32/5

22/2

14/1

1/1

1/1

0/0

1/1

0/0

12/2

6/1

0/0

6/1

4/1

6/1

+Number of time observed/Total number of animals affected.

 

Summary of Gestation Body Weight Values

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Body Weight Values g

0

250.0

13.42

23

254.8

15.54

25

255.2

11.60

22

250.5

11.63

23

3

263.0

15.64

23

267.5

17.55

25

267.6

13.35

22

264.3

10.70

23

6

275.1

15.72

23

278.9

20.02

25

280.4

15.49

22

275.3

12.18

23

9

289.7

18.37

23

294.2

19.78

25

293.8

17.27

22

293.5

12.06

23

12

309.3

18.21

23

312.7

20.96

25

313.9

18.58

22

310.3

14.01

23

15

330.1

19.43

23

334.4

22.24

25

334.7

20.04

22

331.7

16.24

23

18

373.0

20.78

23

379.8

25.04

25

381.0

25.54

22

373.1

17.93

23

20

407.3

24.63

23

417.2

28.57

25

416.9

30.68

22

407.7

21.99

23

N – Number of measures used to calculate mean

SD – Standard Deviation

 

Summary of Gestation Body Weight Change Values

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Body Weight Change Values g

0-3

13.0

4.88

23

12.7

6.00

25

12.5

6.79

22

13.8

5.86

23

3-6

12.1

4.97

23

11/4

6.16

25

12.7

5.82

22

11.0

6.75

23

6-9

14.6

4.26

23

15.3

6.00

25

13.5

6.16

22

18.2

4.03

23

9-12

19.6

4.71

23

18.5

5.40

25

20.0

4.85

22

16.8

5.56

23

12-15

20.9

6.25

23

21.8

5.48

25

20.9

7.34

22

21.4

5.48

23

15-18

42.9

6.61

23

45.3

7.65

25

46.3

9.94

22

41.4

7.41

23

18-20

34.3

7.13

23

37.4

6.02

25

35.9

7.41

22

34.5

7.28

23

0-20

157.4

16.36

23

162.4

19.00

25

161.7

24.30

22

157.2

16.66

23

N – Number of measures used to calculate mean

SD – Standard Deviation

 

Summary of Gestation Food Consumption Values (g/animal/day)

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Food consumption g/animal/day

0-3

16.78

2.350

23

17.09

2.965

25

17.38

2.520

22

18.29

2.163

23

3-6

19.38

1.785

23

20.68

3.265

25

20.21

2.149

22

21.11

2.943

23

6-9

19.59

1.995

23

19.40

2.960

25

20.27

2.752

22

19.78

1.885

23

9-12

21.12

2.002

23

21.44

2.690

25

21.58

3.274

22

21.94

2.560

23

12-15

22.62

2.660

23

22.88

2.874

25

22.88

2.558

22

23.43

2.953

23

15-18

24.87

2.112

23

25.65

3.328

25

26.61

3.223

22

25.94

2.935

23

18-20

23.52

3.113

23

24.42

3.101

25

25.80a

3.073

22

24.83

2.898

23

0-20

21.01

1.578

23

21.51

2.574

25

21.92

2.256

22

22.05

1.913

23

N – Number of measures used to calculate mean                        aSignificantly difference from control; (p<0.05)

SD – Standard Deviation

 

Summary of Maternal and Developmental Observation at Uterine Examination

Endpoint

 

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

No. Females on Study

 

25

25

25

25

No. Not Pregnant

 

2

0

3

2

No. Pregnant

 

23

25

22

23

Pregnancy Index Percent

 

92.0

100.0

88.0

92.0

Corpora Lutea

               No. per Animal

Mean

SD

N

17.3

3.76

23

16.9

2.19

25

16.1

2.51

22

16.4

2.13

23

Implantation Sites

               No. per Animal

Mean

SD

N

14.8

2.09

23

15.5

1.29

25

15.0

3.10

22

14.6

2.76

23

Preimplantation Loss

               % per Animal

Mean

SD

N

12.88

12.640

23

7.12

9.745

25

7.77

13.971

22

10.90

13.569

23

Viable Fetuses

               No. per Animal

Mean

SD

N

13.6

3.00

23

15.0

1.31

25

14.3

3.14

22

13.9

2.61

23

Fetal Sex Ratio

               % Males per Animal

Mean

SD

N

47.0

12.52

23

50.7

11.86

25

49.5

14.51

22

44.0

15.85

23

Post implantation Loss

               % per Animal

Mean

SD

N

8.06

16.797

23

3.53

4.775

25

4.36

7.485

22

4.55

4.995

23

Non-viable Fetuses

               No. per Animal

Mean

SD

N

0.0

0.00

23

0.0

0.00

25

0.0

0.00

22

0.0

0.00

23

Litter Size

               No. per Animal

Mean

SD

N

13.6

3.00

23

15.0

1.31

25

14.3

3.14

22

13.9

2.61

23

Resorption: Early + Late

               No. per Animal

Mean

SD

N

1.2

2.41

23

0.6

0.77

25

0.7

1.13

22

0.7

0.76

23

Resorptions: Early

               No. per Animal

Mean

SD

N

1.2

2.41

23

0.6

0.77

25

0.7

1.13

22

0.7

0.76

23

Resorptions: Late

               No. per Animal

Mean

SD

N

0.0

0.00

23

0.0

0.00

25

0.0

0.00

22

0.0

0.00

23

No. – Number

N – Number of measures use to calculated mean

SD – Standard Deviation

 

Summary of Gravid Uterine Weight and Adjusted Body Weight/Body Weight Change Values

Endpoint

0 mg/kg/day

100 mg/kg/day

300 mg/kg/day

1000 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Gravid Uterine Weight, g

81.4

18.41

23

89.0

10.18

25

87.7

19.08

22

81.9

15.36

23

Final Body Weight, g

407.3

24.63

23

417.2

28.57

25

416.9

30.68

22

407.7

21.99

23

Adjusted Final Body Weight, g

325.9

20.41

23

328.2

24.67

25

329.2

20.31

22

325.7

17.48

23

Adjusted Weight Change from Day 0, g

76.0

13.66

23

73.4

14.07

25

74.1

14.10

22

75.3

11.81

23

N – Number of measures used to calculate mean

SD – Standard Deviation

Applicant's summary and conclusion

Conclusions:
In this prenatal developmental toxicity study, the test article Hatcol® 3346 was administered orally by gastric intubation as a single dose daily to mated female rats from GD 0 to 19. Dose levels of 0, 100, 300, and 1000 mg/kg/day were evaluated. All animals survived to scheduled termination on GD 20. No effects of the test article were observed from maternal clinical findings, gestation body weights or body weight gain, food consumption, macroscopic observations, uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, and skeletal examinations. Thus, the no-observed-effect level (NOEL) for maternal and developmental toxicity with Hatcol® 3346 in this study was 1000 mg/kg/day, the highest dose level evaluated. Likewise, Hatcol® 3346 was not teratogenic in the rat at the dose levels evaluated.
Executive summary:

The study was conducted in accordance with Standard Operating Procedures (SOPs) and the protocol as approved by the Sponsor. The study was based on United States EPA, Office of Prevention, Pesticides, and Toxic Substances, Guideline 870.3700, Prenatal Developmental Toxicity, August 1998, Guideline 414, Prenatal Developmental Toxicity Study, the OECD Guideline for Testing of Chemicals, adopted January 22, 2001, U.S. Department of Agriculture’s (USDA) Animal Welfare Act (9 CFR Parts 1, 2, and 3), and the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011.

 

The study was conducted for Chemtura Corporation to determine the developmental toxicity, including the teratogenic potential, of the test article, Hatcol® 3346. All animals received the vehicle, corn oil, or test article via oral gavage once daily from Gestation Day (GD) 0 to 19. Dose levels of 0, 100, 300, and 1000 mg/kg/day were evaluated.

 

Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination on GD 20. All fetuses were given the appropriate external, visceral, and/or skeletal examination.

Homogeneity of the dosing formulations was confirmed at the low and high concentration levels in Week 1 and concentrations of test formulations used for dosing on Weeks 1 and 3 of study ranged from 100.4 and 103.2% of nominal. No test article was found in control samples.

 

All animals survived to scheduled termination on GD 20. No effects of the test article were observed from maternal clinical findings, gestation body weights or body weight gain, food consumption, macroscopic observations, uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, and skeletal examinations. Thus, the no-observed-effect level (NOEL) for maternal and developmental toxicity with Hatcol® 3346 in this study was 1000 mg/kg/day, the highest dose level evaluated. Likewise, Hatcol® 3346 was not teratogenic in the rat at the dose levels evaluated.