Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 25, 2014 to April 28, 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:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
The test article, Reofos 35, was used as received from the Sponsor, and no adjustment was made for purity.
Date Received: November 20, 2013
Supplier: Chemtura Manufacturing Ltd., Manchester, Great Britain
Amount Received: 2116.1334 mL (1 container)
Label Identification: Reofos 35
Batch Number: 2012123125
Physical Characteristics: Clear, colorless liquid
Retest Date: March 22, 2016
Storage: Room temperature
TMC Number: 130B9E

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
A total of 112 time-mated female CD® [Crl:CD®(SD)] rats (approximately 8 to 10 weeks of age) were received from Charles River Laboratories, Portage, Michigan on February 25 and 26, 2014. The animals were randomly assigned to study and dosing began on GD 0. Prior to the initiation of dose administration, the Study Director reviewed the GD 0 body weight and detailed observation data for all animals and gave final approval for assignment to study.

Randomization, Assignment to Study, and Maintenance
Using a standard, by weight, measured value randomization procedure, 100 female animals (weighing 179 to 254 g, at randomization) were assigned to the control and treatment groups identified in the following table.
Group Assignments
Group Number Dose Level (mg/kg/day) Number of Time-mated Females
1 0 25
2 100 25
3 200 25
4 400 25

Animals assigned to study had body weights within ±20% of the mean body weight. Extra 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. 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:
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, 200, and 400 mg/kg/day at a dose volume of 5 mL/kg/day. 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:
Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see following table) were collected using a positive displacement pipette, while stirring, and placed into amber glass bottles. Following acceptance of the analytical results (signing of the final report) by the Study Director, or at the discretion of the Study Director, backup samples will be discarded. Tabulated data is listed below under "any other information"

Samples were stored refrigerated at 2 to 8°C until shipped on gel packs to the MPI Research, Inc., site at State College, Pennsylvania, for analysis. All analytical work was conducted by MPI Research, Inc., using an analytical method developed by MPI Research, Inc.

Objective: To determine the homogeneity and concentration of Reofos 35 in dosing formulations.
Analytical Method Number and Title: 399-246-A-02 (V0008648-3): Determination of Reofos 35 in Corn Oil. Dose Formulations by GC-FID
Reference Standard: Reofos 35; Lot Number: 2012123125; MPI Research Inventory ID: SP0014320; Storage: Room temperature; Correction Factor: None
Data Collection and Analysis Software: Empower™ 2: Build 2154 (Waters Technologies Corporation®). ExyLIMS Version 3.0 (MPI Research, Mattawan, Michigan)
GC Conditions: Gas chromatography system equipped with a Hewlett Packard HP-1 column, 30 m x 0.25 μm, 0.25 μm thickness with a flame ionization detector.
Analysis Description: Prior to analysis, samples were diluted with 2-propanol (diluent) to within the range of the calibration curve. Vehicle samples were diluted with diluent using a dilution factor of 8. An aliquot of each sample was injected into the GC-FID system for analysis.
Regression Type: Linear unweighted
Study Sample Receipt(s): Number of Samples: 48; Date(s) of receipt by Analytical Department:
February 26, 2014 to March 12, 2014
Study Sample Storage Conditions: Refrigerated (2 to 8°C)
Storage Stability: All samples were analyzed within the established storage stability determined under MPI Research Study Number 399-243.
Analysis Period: March 6, 2014 to March 27, 2014

Run Acceptance Criteria
System Suitability Test Standards:
1. Injection repeatability (peak area and retention time) ≤5% RSD (relative standard deviation)
2. Resolution between the analyte peak and any adjacent peaks must be ≥1.5
3. Tailing Factor (T) ≤2
4. Theoretical Plates (N) ≥2000
Calibration Standards:
1. Accuracy within ±10% of the nominal concentration
2. 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 ±10% of the nominal concentration
Blank Injections: ≤20% of the limit of quantitation (LOQ)

Assessments
Homogeneity:
1. Average concentration within ±10% of the nominal concentration
2. Precision ≤5% RSD
Concentration:
1. Average concentration within ±10% of the nominal concentration
2. Precision ≤5% RSD
3. Vehicle (control) samples < LOQ

Results and Discussion
Conclusion: A total of 36 samples were analyzed for Reofos 35 in dosing formulations using a validated method. The analytical data demonstrated acceptable performance of the method for all reported results. In dataset 030614, there was a programming error in the analytical sequence, which resulted in some samples (Group 1 Week 1 and Group 1 Week 2) not being injected and other samples (Group 2 Week 1) being injected multiple times. All data generated in this dataset is reported; therefore, there are additional replicates for the homogeneity and concentration analyses for the Group 2 Week 1 samples. All samples met the acceptance criteria, so there is no suspected impact.
Deviations: This study phase was conducted in accordance with the protocol with the exception of the following deviations:
Standard Operating Procedure (SOP) Deviations:
SOP deviations were acknowledged by the Study Director and documented in the raw data.
In the opinion of the Study Director, none of the SOP deviations were considered to have affected the quality or integrity of the study.
Details on mating procedure:
Time-mated females used in the study - no details specified in the study report.
Duration of treatment / exposure:
19 days
Frequency of treatment:
Daily
Duration of test:
20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 200, and 400 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Fresh vehicle, corn oil, was dispensed for use on study weekly and was stored refrigerated at 2 to 8°C when not in use.
The test article, Reofos 35, was used as received from the Sponsor, and no adjustment was made for purity. Formulations of the test article were prepared weekly at nominal concentrations of 20, 40, and 80 mg/mL, and were stored refrigerated at 2 to 8°C when not in use.

Analysis of Dosing Formulations
Dosing formulations prepared for the study were evaluated for homogeneity and concentration. Appropriate samples (see table in Any other information) were collected using a positive displacement pipette, while stirring, and placed into amber glass bottles. Following acceptance of the analytical results (signing of the final report) by the Study Director, or at the discretion of the Study Director, backup samples will be discarded.
Stability has been established for at least 10 days under refrigerated (2 to 8°C) conditions under MPI Research, Inc., Study Number 399-246.

Justification for Route of Administration
The oral route is one of the potential routes of human exposure to this test article.

Justification of Dose Levels
The dose levels were selected by the Sponsor on the basis of available data from a recently conducted pilot prenatal developmental toxicity study (MPI Research, Inc., Study Number 399-245) and available data from a previously conducted reproduction/developmental screening study that included Reofos 35 (MPI Research, Inc., Study Number 1038-004). In the pilot study (MPI Research, Inc., Study Number 399-245), mortality (60%), lower body weights and body weight gain, lower food consumption, and reduced fetal body weights were observed in time-mated females treated daily from GD 0 through GD 20 with Reofos 35 at 500 mg/kg/day.
In the screening study (MPI Research, Inc., Study Number 1038-004), animals were treated daily for 8 weeks with Reofos 35 at 400 mg/kg/day. Clinical findings (salivation), lower body weights and body weight gain, reproductive effects, organ weight changes, and microscopic observations were observed.
Based on the available data, dose levels up to 400 mg/kg/day were evaluated in time-mated females from GD 0 to 20. The low- and mid-dose are at approximate log intervals and were chosen to provide a graded response.

Examinations

Maternal examinations:
In-life Examinations
Cage-side Observations: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily. On occasion, veterinary consultations were conducted during the course of the study. All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.
Detailed Clinical Observations: Daily from GD 0 through 20 (60 to 90 minutes post dose on dosing days), each animal was removed from the cage and given a detailed clinical examination. On occasion, clinical observations were recorded at unscheduled intervals. 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, respiratory and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior.
Body Weights and Body Weight Changes: Body 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 Consumption: Food consumption was measured and recorded on the corresponding body weight days and calculated for the same.

Postmortem Study Evaluations
Maternal Necropsy: A complete necropsy was performed on all dams 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:
Ovarian and Uterine Examination
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. Dams had uterine examinations conducted without knowledge of the treatment group. 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 sites. If no foci were detected, the female was considered to be non-pregnant.
Fetal examinations:
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. Fetal external, visceral, and skeletal examinations were conducted without knowledge of the treatment group.
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 technique. 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 Dawson 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.
These artifacts are not reported, but are maintained in the study data.
Statistics:
See Any other information for Statistics data.
Indices:
Fetal and litter incidences are reported, but only the litter incidences were statistically analyzed.
Historical control data:
Historical Control Developmental Toxicity Data
Sprague Dawley Rat
07/2006 to 07/2011
Number of Studies: 50
Number of Litters Evaluated: 1230
Number of Fetuses Evaluated – External: 14180
Number of Fetuses Evaluated – Visceral: 7072
Number of Fetuses Evaluated – Skeletal: 7106

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. It was observed at least once in 4, 8 and 15 animals in the 100, 200, and 400 mg/kg/day dose groups, respectively. Its occurrence on study was likely a pharmacologic response to the test article and was not considered adverse. Other clinical findings observed at dose levels ≤200 mg/kg/day occurred at low incidence or with similar frequency as controls and considered unrelated to the test article. In the 400 mg/kg/day dose group, additional clinical findings observed within the first several days of dosing (GD 1 to 3) and considered test article related included decreased activity (one animal), red material around the nose/mouth (nine animals), hunched posture (one animal), thin appearance (two animals), hair on the face discolored brown (two animals), and hair on the forelimbs discolored brown (two animals). These clinical findings were transient and were not observed among the 400 mg/kg/day animals for the remainder of the study.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 400 mg/kg/day dose group, a mean body weight gain of 0.1 g over GD 0 to 3 differed statistically from the mean weight gain of 14.0 g in the control group. This low weight gain in the 400 mg/kg/day dose group was considered test article related and adverse correlating with a period of low food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 200 mg/kg/day dose group, mean food consumption throughout gestation was higher relative to mean control values and these differences were statistically significant over GD 3 to 6 (+23%), GD 6 to 9 (+18%), GD 9 to 12 (+17%), GD 12 to 15 (+10%), GD 18 to 20 (+10%), and GD 0 to 20 (+13%). The increase in food consumption in the 200 mg/kg/day dose group may have been test article related but the effect was not considered adverse and only over GD 0 to 20 did it correlate with statistically higher body weight gain relative to controls. In the 400 mg/kg/day dose group, mean food consumption over GD 0 to 3 at 9.1 g/day was statistically lower than the mean control value of 13.6 g/day. This was considered test article related and adverse correlating with a period of statistically lower body weight gain.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: red or white foci and swollen mucosa of the nonglandular portion of the stomach

Details on maternal toxic effects:
In-life Examinations
Mortality
All animals survived to scheduled termination on GD 20.

Detailed Clinical Observations
Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. It was observed at least once in 4, 8 and 15 animals in the 100, 200, and 400 mg/kg/day dose groups, respectively. Its occurrence on study was likely a pharmacologic response to the test article and was not considered adverse. Other clinical findings observed at dose levels ≤200 mg/kg/day occurred at low incidence or with similar frequency as controls and considered unrelated to the test article. In the 400 mg/kg/day dose group, additional clinical findings observed within the first several days of dosing (GD 1 to 3) and considered test article related included decreased activity (one animal), red material around the nose/mouth (nine animals), hunched posture (one animal), thin appearance (two animals), hair on the face discolored brown (two animals), and hair on the forelimbs discolored brown (two animals). These clinical findings were transient and were not observed among the 400 mg/kg/day animals for the remainder of the study.

Body Weights and Body Weight Changes
No test article effect at the 100 and 200 mg/kg/day dose levels was observed on gestation body weights or body weight gain. A statistically significant increase in mean weight gain over GD 0 to 20 in the 200 mg/kg/day dose group relative to the control mean value (170.7 g vs.156.7 g in controls) was considered unrelated to the test article but did correlate with statistically higher food consumption relative to the controls over this period. In the 400 mg/kg/day dose group, a mean body weight gain of 0.1 g over GD 0 to 3 differed statistically from the mean weight gain of 14.0 g in the control group. This low weight gain in the 400 mg/kg/day dose group was considered test article related and adverse correlating with a period of low food consumption. However, the effect was transient and over the ensuing intervals of GD 3 to 6 and GD 6 to 9, mean body weight gains in the 400 mg/kg/day dose group of 22.7 g and 22.8 g were statistically higher than the mean body weight gains of 15.2 g (49% higher) and 19.5 g (17% higher) respectively, in the control group. For the remainder of gestation and over the entire GD 0 to 20 period, mean weight gains in the 400 mg/kg/day dose group were comparable to mean control values.

Food Consumption
No adverse effect of test article at the 100 and 200 mg/kg/day dose levels was observed on gestation food consumption. Mean food consumption throughout gestation in the 100 mg/kg/day dose group was comparable to mean control values. In the 200 mg/kg/day dose group, mean food consumption throughout gestation was higher relative to mean control values and these differences were statistically significant over GD 3 to 6 (+23%), GD 6 to 9 (+18%), GD 9 to 12 (+17%), GD 12 to 15 (+10%), GD 18 to 20 (+10%), and GD 0 to 20 (+13%). The increase in food consumption in the 200 mg/kg/day dose group may have been test article related but the effect was not considered adverse and only over GD 0 to 20 did it correlate with statistically higher body weight gain relative to controls. In the 400 mg/kg/day dose group, mean food consumption over GD 0 to 3 at 9.1 g/day was statistically lower than the mean control value of 13.6 g/day. This was considered test article related and adverse correlating with a period of statistically lower body weight gain. The effect on food consumption at 400 mg/kg/day was transient and for the remainder of gestation, mean food consumption in this group was 4% to 17% higher than the mean control values with statistical significance identified over GD 3 to 6 (+15%), GD 9 to 12 (+17%), and GD 12 to 15 (+13%).

Postmortem Study Evaluations
Uterine and Ovarian Examinations
The pregnancy indices were 96%, 96%, 96%, and 92% in the control, 100, 200, and 400 mg/kg/day dose groups providing 24, 24, 24, and 23 GD 20 litters with fetuses for evaluation, respectively. No test article effect was observed from GD 20 uterine implantation data. In the 100 mg/kg/day dose group, the mean pre-implantation loss index was 14.63% and statistically higher than the 5.01% in controls but in the absence of a similar increase in this index at the higher dose levels, this was considered incidental and unrelated to the test article. All other mean uterine implantation parameters (corpora lutea, implantation sites, viable fetuses, post-implantation loss, litter size, and resorption sites [early, late, and total]) in the treated groups were comparable to mean control values. Mean gravid uterine weights in the 100 and 400 mg/kg/day dose groups were comparable to mean control values. In the 200 mg/kg/day dose group, the mean gravid uterine weight at 80.26 g was statistically higher than the mean control value of 71.24 g. This was considered related to the slightly greater number of fetuses in utero, and unrelated to the test article. The mean adjusted GD 20 body weights (GD 20 body weight minus the gravid uterine weight) and adjusted body weight change GD 0 to 20 in the treated groups were comparable to mean control values.

Maternal Macroscopic Observations
No test article effect at 100 and 200 mg/kg/day was observed from the maternal macroscopic examinations. Macroscopic findings observed among these animals occurred at low incidence (single animals affected), and were considered incidental and unrelated to the test article. In the 400 mg/kg/day dose group, red or white foci and swollen mucosa of the nonglandular portion of the stomach were observed in 3/25 animals. Their occurrence in the 400 mg/kg/day group was considered test article related as similar findings were not observed among control animals or animals at the lower dose levels. No other macroscopic findings were observed among the 400 mg/kg/day animals.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
400 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):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Fetal Evaluations
Fetal Sex Ratio
No test article effect was observed from fetal sex ratios (% male fetuses/animal). Mean fetal sex ratios in the treated groups ranged from 45.4% to 53.4% and were comparable to the mean of 54.0% in the control group.
Fetal Body Weights
No test article effect was observed on fetal body weights. In the 100 mg/kg/day dose group, fetal body weights, distinguished by sex and for the combined sexes, were comparable to mean control values. In the 200 and 400 mg/kg/day dose groups, mean fetal body weights were higher than mean control values and although the differences were slight (5% to 7%) in most instances they were statistically significant and outside the ranges of recent historical control data for the laboratory. The increase in mean fetal body weights observed in the 200 and 400 mg/kg/day dose groups although statistically significant relative to control values, were still relatively slight 5 to 7% and not considered adverse or indicative of a test article-related response.
External Examinations
No test article effect was observed from the fetal external examinations. No malformations or developmental variations were observed among the control or treated fetuses from the external examinations.
Visceral Examinations
No test article effect was observed from the fetal visceral examinations. No visceral malformations were observed among the control or treated fetuses. The only visceral developmental variation observed was dilated ureter. This variation has a high historical presence in this laboratory (Appendix O, maximum litter incidence of 20.8%) and its occurrence in a single fetus in the 400 mg/kg/day dose group (litter incidence 4.3%) was considered incidental and unrelated to the test article.
Skeletal Examination
No test article effect was observed from the fetal skeletal examinations. The few skeletal malformations observed among fetuses in the treated groups (one fetus in the 200 mg/kg/day dose group and two fetuses from a single litter in the 400 mg/kg/day dose group) were dissimilar in appearance and considered unrelated to the test article. No skeletal malformations were observed in fetuses from the 100 mg/kg/day dose group; one control fetus had a skeletal malformation. 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 on skeletal development was indicated from these data.

Effect levels (fetuses)

Dose descriptor:
other: Not determined
Remarks on result:
not measured/tested

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

 Homogeneity

Analysis of the low- and high-dose formulations (20.0 and 80.0 mg/mL, respectively) used for dosing the first week of study confirmed they were homogeneous as prepared and therefore met the laboratory’s acceptance criteria of 100±10% of nominal, and percent relative standard deviation (RSD) ≤5. These analytical results are summarized in the table below.

Homogeneity

Dose Level (mg/kg/day)

Nominal Concentration (mg/mL)

Average Calculated Concentration (mg/mL)

Average %Recovery

%Relative Standard Deviationc

100

400

20.0

80.0

18.7372a

81.6908b

93.7a

102.1b

1.9

1.5

aRepresents an average of seven samples from the mixing container (two top, two middle and three bottom. Samples were collected while the formulation was stirring.

bRepresents an average of six samples from the mixing containers (two top, two middle and two bottom). Samples were collected while the formulation was stirring.

cAverage %recovery was calculated from the nominal concentration.

Concentration

Mean concentrations of formulations used for dosing each of the three weeks of study ranged between 93.2 and 102.1% of nominal confirming that animals were receiving the appropriate dose levels when the dose was administered at 5 mL/kg. No test article was found in the control samples. These results are summarized in the table below.

Concentration

Dose Level (mg/kg/day)

Nominal Concentration (mg/mL)

Average Calculated Concentrationa(mg/mL)

Average %Recoverya,b

0

100

200

400

0.0

20.0

40.0

80.0

BLQ

18.6350 – 19.1496

37.4113 – 38.8650

81.0502 – 81.6550

NA

93.2 – 95.7

93.5 – 97.2

101.3 – 102.1

aResults are the range of values determined during Weeks 1, 2 and 3.

bAverage %recovery was calculated from the nominal concentration.

BLQ – Below the Limit of Quantification (<0.4 mg/mL)

NA – Not Applicable

 

Summary of Gestation Detailed Clinical Observations+

Days 0 to 20

Observation

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

Number of Animals Observed

25

25

25

25

Behavior/Activity

               Activity decreased

               Salivation

 

0/0

0/0

 

0/0

6/4

 

0/0

36/8

 

2/1

85/15

External Appearance

               Discharge, Red, Anogenital region

               Ear/portion of ear missing, Ear/left

               Material around mouth, Red

               Material around nose, Red

               Posture hunched

               Thin

 

0/0

6/1

0/0

0/0

0/0

0/0

 

0/0

0/0

0/0

0/0

0/0

0/0

 

1/1

5/1

0/0

0/0

0/0

0/0

 

0/0

0/0

1/1

11/9

2/1

3/2

Pelage/Skin

               Hair, discolored, Brown, Face

               Hair, discolored, Brown, Forelimb/left

               Hair, discolored, Brown, Forelimb/right

               Hair, discolored, Black, Thoracic region

               Hair, sparse, Abdominal region

               Hair, sparse, Cervical region

               Hair, sparse, Forefoot/left

               Hair, sparse, Forefoot/right

               Hair, sparse, Forelimb/left

               Hair, sparse, Forelimb/right

               Hair, sparse, Hind foot/left

               Hair, sparse, Hind foot/right

               Hair, sparse, Hind limb/left

               Hair, sparse, Hind limb/right

               Hair, sparse, Inguinal region/left

               Hair, sparse, Lumbar region

               Hair wet, Anogenital region

               Scabbed area, Cervical region

               Scabbed area, Forelimb/right

 

0/0

0/0

0/0

0/0

14/1

0/0

47/7

42/6

1/1

5/1

9/2

9/2

7/1

7/1

0/0

1/1

0/0

0/0

0/0

 

0/0

0/0

0/0

0/0

0/0

0/0

10/3

19/4

12/1

12/1

4/1

4/1

0/0

0/0

6/1

9/1

0/0

2/1

0/0

 

0/0

0/0

0/0

0/0

0/0

30/3

20/2

23/4

54/7

42/5

0/0

0/0

6/1

10/1

0/0

5/1

0/0

0/0

8/1

 

4/2

3/2

3/2

1/1

21/3

0/0

1/1

5/1

35/3

14/1

0/0

0/0

27/3

14/1

0/0

9/1

2/1

0/0

6/2

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

 

Summary of Gestation Body Weight Values

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Body Weight Values g

 

 

 

 

 

 

 

 

 

 

 

 

 

0

214.1

18.94

24

214.8

18.47

24

215.3

20.20

24

215.0

20.55

23

3

228.2

17.73

24

230.3

17.49

24

231.4

20.78

24

215.2

20.71

23

6

243.3

18.60

24

245.3

18.44

24

248.9

22.21

24

237.9

21.35

23

9

262.9

19.04

24

265.9

18.52

24

270.5

23.94

24

260.7

21.19

23

12

283.3

20.42

24

285.6

18.85

24

292.8

27.78

24

284.6

22.81

23

15

301.1

20.34

24

304.3

18.85

24

313.6

29.01

24

303.9

25.00

23

18

336.2

26.04

24

338.9

21.33

24

350.7

33.29

24

336.3

27.33

23

20

370.8

30.31

24

372.3

23.66

24

386.0

36.84

24

368.8

28.85

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

200 mg/kg/day

400 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Body Weight Values g

 

 

 

 

 

 

 

 

 

 

 

 

 

0 – 3

14.0

4.47

24

15.5

5.49

24

16.1

8.51

24

0.1b

9.69

23

3 – 6

15.2

4.09

24

15.0

4.91

24

17.5

4.27

24

22.7b

5.86

23

6 – 9

19.5

4.67

24

20.6

3.86

24

21.6

5.24

24

22.8a

4.01

23

9 – 12

20.4

4.78

24

19.7

4.70

24

22.4

5.45

24

23.8

4.77

23

12 – 15

17.8

5.30

24

18.7

3.29

24

20.8

4.29

24

19.3

4.47

23

15 – 18

35.1

7.86

24

34.7

6.49

24

37.1

5.99

24

32.4

4.75

23

18 - 20

34.6

6.70

24

33.3

5.62

24

35.3

5.29

24

32.5

4.50

23

0 - 20

156.7

19.12

24

157.5

17.40

24

170.7a

21.25

24

153.7

18.35

23

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

SD – Standard Deviation                                                   bSignificantly different from control; (p<0.01)

 

Summary of Gestation Food Consumption Values

Endpoint

Study Interval (Day)

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

Mean

SD

N

Mean

SD

N

Mean

SD

N

Mean

SD

N

Food Consumption g/animal/day

 

 

 

 

 

 

 

 

 

 

 

 

 

0 – 3

13.6

1.89

24

13.8

2.13

24

14.2

3.48

24

9.1b

3.23

23

3 – 6

17.1

2.31

24

19.2

1.73

24

21.1b

2.52

24

19.6a

4.92

23

6 – 9

18.5

2.27

24

19.4

1.43

24

21.9b

3.30

24

20.5

4.27

23

9 – 12

19.1

2.15

24

20.1

1.64

24

22.4b

2.87

24

22.3b

2.66

23

12 – 15

22.3

2.04

24

23.3

1.56

24

24.6b

3.03

24

25.3b

3.22

23

15 – 18

24.5

3.17

24

25.1

2.21

24

26.6

3.32

24

25.6

3.17

23

18 - 20

23.1

3.35

24

24.8

2.12

24

25.4a

3.56

24

24.2

2.85

23

0 - 20

19.6

1.97

24

20.6

1.34

24

22.2b

2.66

24

20.8

2.70

23

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

SD – Standard Deviation                                                   bSignificantly different from control; (p<0.01)

 

Summary of Maternal Developmental Observations at Uterine Examination

Endpoint

 

0 mg/kg/day

100 mg/kg/day

200 mg/kg/day

400 mg/kg/day

No. Females on Study

25

25

25

25

No. Not Pregnant

1

1

1

2

No. Pregnant

24

24

24

23

Pregnancy Index Percent

96.0

96.0

96.0

92.0

No. Females with Viable Fetuses Day 20 Gestation

24

24

24

23

Corpora Lutea

               No. per Animal

Mean

SD

N

12.9

2.08

24

13.5

2.08

24

13.5

1.53

24

13.8

2.84

23

Implantation Sites

               No. per Animal

Mean

SD

N

12.3

2.09

24

11.3

1.57

24

12.7

1.95

24

12.3

1.42

23

Preimplantation Loss

               % per Animal

Mean

SD

N

5.01

7.506

24

14.36b

13.246

24

5.67

7.263

24

9.51

11.254

23

Viable Fetuses

               No. per Animal

Mean

SD

N

11.5

2.04

24

10.8

1.84

24

12.0

1.43

24

11.4

1.50

23

Fetal Sex Ratio

               % Males per Animal

Mean

SD

N

54.0

16.36

24

49.8

15.02

24

53.4

16.60

24

45.4

18.37

23

Postimplantation Loss

               % per Animal

Mean

SD

N

6.27

7.070

24

4.73

7.509

24

4.94

7.831

24

6.79

5.383

23

Nonviable Fetuses

               No. per Animal

Mean

SD

N

0.0

0.00

24

0.0

0.00

24

0.0

0.00

24

0.0

0.00

23

Litter Size

               No. per Animal

Mean

SD

N

11.5

2.04

24

10.8

1.84

24

12.0

1.43

24

11.4

1.50

23

Resorptions: Early + Later

               No. per Animal

Mean

SD

N

0.8

0.88

24

0.5

0.78

24

0.6

1.01

24

0.8

0.65

23

Resorptions: Early

               No. per Animal

Mean

SD

N

0.8

0.85

24

0.5

0.78

24

0.6

0.97

24

0.8

0.65

23

Resorptions: Late

               No. per Animal

Mean

SD

N

0.0

0.00

24

0.0

0.00

24

0.0

0.00

24

0.0

0.00

23

No. – Number                                                                                      bSignificantly different from control; (p<0.01)

N – Number of measured used to calculate mean

SD – Standard Deviation

Applicant's summary and conclusion

Conclusions:
This study was conducted to evaluate the potential adverse effects of the test article, Reofos 35, on systemic toxicity, female reproductive performance, and development of the fetus following repeated exposure to pregnant rats. Dose levels evaluated were 0 (corn oil vehicle), 100, 200, or 400 mg/kg/day and animals were treated orally with a single dose daily from GD 0 to 19. All animals survived to scheduled termination. The No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed-Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. Reofos 35 was not teratogenic in the rat at the dose levels tested.
Executive summary:

The study was conducted for Chemtura Corporation to evaluate the potential adverse effects of the test article, Reofos 35, following repeated exposure to pregnant animals from Gestation Day (GD) 0 to 19, including systemic toxicity, female reproductive performance, and evaluation of the fetuses.

The study was conducted in accordance with Standard Operating Procedures (SOPs) and the protocol as approved by the Sponsor. All SOP deviations were acknowledged by the Study Director and documented in the raw data. In the opinion of the Study Director, none of the SOP deviations affected the quality or integrity of the study. This study was based on Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011, the United States EPA, Office of Prevention, Pesticides, and Toxic Substances, Guideline 870.3700, Prenatal Developmental Toxicity, August 1998, and the OECD 414 Guideline for the Testing of Chemicals, January 2001.

 

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-dose levels in Week 1 and concentrations of test formulations used for dosing study ranged from 93.2 to 102.1% of nominal. No test article was found in control samples.

All control and treated animals survived to scheduled termination. Salivation was observed in each of the Reofos 35-treated groups and considered a test article-related effect. Its occurrence was attributed to a pharmacologic response to the test article and was not considered adverse. At dose levels ≤200 mg/kg/day, no adverse effects of the test article were observed from maternal clinical findings, gestation body weights, body weight change, food consumption, or macroscopic examinations. At 400 mg/kg/day, maternal toxicity was restricted to early in the treatment period (GD 0 to 3) and included clinical findings (i.e., decreased activity, red material around the nose/mouth, hunched posture, thin appearance, hair on the face discolored brown, and hair on the forelimbs discolored brown), low gestation weight gain, and low food consumption. No adverse effect of treatment with Reofos 35 at the dose levels evaluated was observed on pregnancy indices, uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, or skeletal evaluations. In the 400 mg/kg/day dose group, a low incidence of red or white foci and swollen mucosa of the nonglandular portion of the stomach was observed in the maternal macroscopic evaluations and considered test article-related.

Thus, in this rat oral prenatal developmental toxicity study with Reofos 35, the No-Observed-Adverse-Effect Level (NOAEL) for maternal toxicity was 200 mg/kg/day and the No-Observed–Effect Level (NOEL) for developmental toxicity was 400 mg/kg/day, the highest dose level evaluated. Reofos 35 was not teratogenic in the rat at the dose levels tested.