Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: Combined Oral Repeated Dose and Reproductive/Developmental Toxicity Screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 11 2003 to October 2 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, carried out according to recognised guideline.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Phosflex TXP was administered in a vehicle of corn oil by oral gavage at dose levels of 0 (vehicle control), 25, 200 or 1000 mg/kg/day to 11 Sprague-Dawley rats/sex/group for the assessment of reproductive and developmental toxicity, and target organ toxicity. Rats were dosed 7 days/week for 2 weeks prior to mating, during the 2-week mating period, and throughout gestation and lactation until sacrifice, for a total of approximately 33 days of dosing for males and 48 days for females. Doses were administered at a constant volume of 2 ml/kg. The control and high dose groups included an additional 5 rats/sex designated as recovery animals. During the recovery phase of the study, the protocol was amended to include a mating of the recovery animals as well as a cross-over mating. The cross-over mating consisted of cohabitation of high dose males with naive females, following which recovery animals were mated within their respective groups. The mating of recovery animals was added to the study in order to determine whether the apparent adverse effects of test material on reproductive function were reversible, whereas cross-over mating was used to aid in determining whether the observed effects were male- or female-mediated. Recovery males were held for 4 weeks after dosing cessation prior to conducting the cross-over mating, while recovery females were held for 3 weeks prior to performing the within-group mating. Recovery males were euthanized and necropsied at the end of the mating trials (after 7.5 weeks) and recovery females were necropsied after postnatal day 4 (9 weeks of recovery). Parental food consumption, body weight, body weight gain, reproductive performance, organ weight and histopathology were evaluated during the study, along with fetal body weights and survival.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Test Animals
Sixty male and 60 nulliparous female Sprague-Dawley rats were purchased from Taconic Farms, Germantown, NY for use in this study and were received on June 5, 2003. The rats were approximately 7 weeks old; males weighed 161-191 grams on receipt, while females weighed 136-166 grams. Rats were approximately 8 weeks old at initiation of dosing. Each rat was identified by a unique ear tag; cage cards were marked with both study and animal numbers.

Food and Water
Harlan Teklad Certified Rodent Diet 8728C was provided ad libitum, except during scheduled fasting periods. Each lot was analyzed for contaminants to ensure that none were present at concentrations that would be expected to interfere with the conduct of the study. The analytical data from diet lots used in the study were reviewed by the facility veterinarian and maintained in a central file at the testing facility. City of Chicago water was provided ad libitum to all animals by an automatic watering system. Supply water was analyzed for contaminants as defined by the U.S. EPA "National Primary Drinking Water Regulations" (Title 40, Code of Federal Regulations, Part 141). Water analysis records are retained on file at the testing facility. No contaminants were known to be present in the food or water that would have interfered with the interpretation of the study.

Housing, and Environment
Upon arrival, rats were individually housed in stainless steel cages equipped with an automatic watering system. During the mating period, one female and one male were housed together in polycarbonate shoebox cages with corncob bedding and an automatic watering system. When evidence of mating was observed, the male was returned to its home cage, while the female remained in the shoebox cage. To the best of our knowledge, no known contaminants were present in the bedding that would have confounded the interpretation of the study.
Animals were housed in accordance with the Guide for the Care and Use of Laboratory Animals (1996). During the study, the animal room(s) temperature and relative humidity were maintained to avoid extreme fluctuations from the protocol-specified ranges of 64-79°F and 30-70%, respectively. Controls were set to maintain the animal rooms at a minimum of 10 air changes per hour. Fluorescent lighting in the animal rooms was on a 12-hour light/dark cycle. The housing and environmental conditions in the animal rooms were in general agreement with the Guide for the Care and Use of Laboratory Animals (1996).
Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
corn oil
Details on exposure:
The test material dosing formulations were prepared approximately every 4 weeks by Experimur at graded concentrations of 1.25%, 10%, and 50%, which corresponded to target doses of 25, 200, and 1000 mg/kg/day, when administered at a dosing volume of 2 ml/kg. Dose levels of the test material were selected by the Sponsor based on findings from previous toxicity studies with the test material.

The core parental treatment and control groups consisted of 11 males and females/group. Animals were administered the test material or vehicle by gavage, 7 days/week, for 2 weeks prior to mating. Treatment began at approximately 8 weeks of age for both males and females. At the end of the 2-week treatment period, one male was housed with one female of the same dose level and allowed to mate for up to 2 weeks.

Details on mating procedure:
The administration of test material continued during the mating period. Daily vaginal smears, taken from female rats during the mating period, were evaluated for the presence of sperm and the stage of estrus cycle. The presence of sperm in the smear or a sperm-plug indicated a positive mating, and that day was designated as gestation Day 0. Up to 11 sperm-positive female rats per group were selected to produce the Fl generation. Dams selected for littering were allowed a natural parturition with two daily checks that began on gestation Day 18. Administration of the test material continued through gestation and lactation until postpartum Day 4; in effect, dams were dosed until sacrificed. All parental males were dosed for a minimum of 33 consecutive days prior to sacrifice and were terminated when they were no longer needed for mating.

The original protocol specified that the control and high-dose groups would each include 5 additional rats/sex to be treated for the same duration but not mated. These rats were designated as recovery animals and were to be held for 14 days after treatment termination to evaluate the recovery potential from any adverse treatment-related effects. However, an amendment to the study protocol was added when only two of the females treated with 200 mg/kg/day and none of the females treated with 1000 mg/kg/day underwent successful parturition. The amended protocol required mating of the high-dose recovery males with naive untreated females (cross-over mating); following the cross¬over mating, recovery animals were mated within their respective dose levels. The mating of recovery animals was intended to help determine the reversibility, within 4 weeks, of apparent adverse effects on reproductive function. Cross-over mating was expected to help determine whether the effects were male- or female-mediated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Phosflex TXP in corn oil was measured using high pressure liquid chromatography (HPLC) with UV detection. Aliquots of the dosing formulations were diluted and analyzed with detection at 254 nm. Analyses followed a study-specific procedure approved by the Study Director. Stability was initially determined during a range-finding study.

Materials and Methods
Standards were prepared by accurately weighing a known amount of Phosflex TXP and diluting to volume with mobile phase in a volumetric flask.
The HPLC conditions were:

Column: Phenomonex Lichosphere RP 18e 5^ 4.0 x 250 mm
Mobile Phase: 80/20 Acetonitrile/methanol
Flowrate: 1.0ml/min
Detector: UV at 254 nm
Injection volume: 20 ul
Retention time: approx. 3.9 minutes

Phosflex TXP is a mixture which was quantitated by fusing all major isomers into a single peak and setting the total area equal to the calibration amount.

An external standard calibration was performed by running known concentrations of Phosflex TXP to establish a standard curve. Each day that samples were analyzed, the instrument was recalibrated.

Dosing formulations, in a vehicle of com oil, were analyzed by removing an aliquot and weighing it in a volumetric flask. The flask was diluted with ethyl acetate, placed in an LC vial and analyzed. Each dosing formulation was analyzed in duplicate by testing two separate samples.

The concentration and homogeneity of the low and high dosing formulations were determined by analysis of samples collected at the top, middle and bottom of the flask. The concentration of the mid-dose formulation was determined from samples collected at the middle of the flask. Duplicate samples were collected from each location.

A 500 mg/ml solution of Phosflex TXP in com oil was prepared during the method validation phase and a single sample was periodically analyzed for stability for up to 63 days.

Results
The dosing formulations were homogeneous and ranged between 2-6% of target concentrations (Exhibit A). Minimum differences in concentration were noted between samples collected at the top, middle or bottom of the flask. The Phosflex TXP formulation proved to be stable for more than 7 weeks, with differences in the analytically-determined concentrations ranging from 0.7% to -5.9% compared to the initial concentration (Exhibit B).

Conclusion

Analysis was conducted on samples of the dosing formulations collected from each dose level of all preparations. The dosing formulations were homogeneous. The analytically-determined concentrations met the acceptance criteria for solution formulations as specified in the study protocol (i.e., they were within 10% of the target concentrations). In the case of homogeneity analyses, each set of values was also within 10% of each other. Stability of Phosflex TXP in corn oil was determined to be greater than 7 weeks.
Duration of treatment / exposure:
All rats were administered the test material or vehicle by oral gavage, 7 days/week, for 2 weeks prior to mating, and during the 2-week cohabitation period. Females were treated through gestation, lactation and until postpartum Day 4, for a minimum of 48 doses, while males received a minimum of 33 doses. A dosing volume of 2 ml/kg was used in all cases.
Frequency of treatment:
Daily as above.
Details on study schedule:
The administration of test material continued during the mating period. Daily vaginal smears, taken from female rats during the mating period, were evaluated for the presence of sperm and the stage of estrus cycle. The presence of sperm in the smear or a sperm-plug indicated a positive mating, and that day was designated as gestation Day 0. Up to 11 sperm-positive female rats per group were selected to produce the Fl generation. Dams selected for littering were allowed a natural parturition with two daily checks that began on gestation Day 18. Administration of the test material continued through gestation and lactation until postpartum Day 4; in effect, dams were dosed until sacrificed. All parental males were dosed for a minimum of 33 consecutive days prior to sacrifice and were terminated when they were no longer needed for mating.
See below for tabulated data detailing a summary of the study design:
Remarks:
Doses / Concentrations:
25 mg/kg/day
Basis:
actual ingested
administered at a dosing volume of 2 ml/kg
Remarks:
Doses / Concentrations:
200 mg/kg/day
Basis:
actual ingested
administered at a dosing volume of 2 ml/kg
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
administered at a dosing volume of 2 ml/kg
No. of animals per sex per dose:
11 males / females per group
Control animals:
yes, concurrent vehicle
Details on study design:
The core parental treatment and control groups consisted of 11 males and females/group. Animals were administered the test material or vehicle by gavage, 7 days per week, for 2 weeks prior to mating. Treatment began at approximately 8 weeks of age for both males and females. At the end of the two week treatment period, one male was housed with one female of the same dose level and allowed to mate for up to 2 weeks. The administration of test material continued during the mating period. Daily vaginal smears, taken from the female rats during the mating period, were evaluted for the presence of sperm and the stage of the estrus cycle. The presence of sperm in the smear or a sperm plug indicated a positive mating and that day was designated as gestation Day 0. Up to 11 sperm-positive female rats were selected to produce the F1 generation. Dams selected for littering were allowed a natural parturition with two daily checks that began on Day 18. Administration of the test material continued through gestation and lactation until post-partum day 4; in effect, dams were dosed until sacrificed. All parental males were dosed for a minimum of 33 consecutive days prior to sacrifice and were terminated when they were no longer needed for mating.
The original protocol specified that the control and high-dose groups would each include 5 additional rats/sex to be treated for the same duration but not mated. These rats were designated as recovery animals and were to be held for 14 days after treatment termination to evaluate the recovery potential from any adverse treatment-related effects. However, an amendment to the study protocol was added when only two of the females treated with 200 mg/kg/day and none of the females treated with 1000 mg/kg/day underwent sucessful parturition. The amended protocol required mating of the high-dose recovery males with naive untreated females (cross-over mating); floolowing the cross-over mating, the recovery animals were mated within their respective dose levels. The mating of recovery animals was intended to help determine the reversibility, within 4 weeks, of apparent adverse effects on reproductive function. Cross-over mating was expected to help determine whether the effects were male ot female mediated.
Positive control:
None
Parental animals: Observations and examinations:
During the conduct of the test, each rat was evaluated upon dosing for general appearance, as well as overt signs of toxicity; abnormal clinical signs were noted, if observed.
Five rats/sex/group were fasted overnight for blood collection prior to scheduled necropsy. Blood for clinical chemistry and hematology was collected from the abdominal aorta immediately prior to the scheduled necropsy. For dams, the blood was collected from animals in the same or similar physiological state, (i.e. postpartum).

Moribunditv/Mortality Observation: During quarantine, the animals were observed at least once daily for moribundity and mortality. During the conduct of the test, each rat was evaluated upon dosing for general appearance, as well as overt signs of toxicity; abnormal clinical signs were noted, if observed.

Clinical Observations: Detailed clinical examinations were performed on all animals during quarantine, and a hand-held clinical observation was performed weekly on each animal. In conjunction with one of the daily moribundity/mortality checks (G.3.), a cage-side observation was performed daily; pertinent behavioral changes, signs of difficult or prolonged parturition and/or overt signs of toxicity were documented.
Body Weights: The parental rats were weighed during quarantine, and those weights were used for randomization. Parental male rats were weighed 1 day prior to treatment initiation and weekly thereafter. Females were not weighed during cohabitation. Pregnant females were weighed on gestation Days 0, 7, 14, and 21. Dams and their litters were weighed on the day of the observed birth (postnatal Day 0) and on postnatal Day 4. A fasted body weight was collected prior to terminal sacrifice. The fasted body weight was used to calculate organ-to-body weight ratios.

Neurotoxicity Assessment: A functional observational battery (FOB) and automated motor activity assessment were performed toward the end of the study on 5 rats/sex/group (males after 24-28 doses and females after parturition). Motor activity was evaluated using an automated system (Coulbourn True Scan 99®); the evaluation consisted of monitoring activity in an open field over a period 21 minutes. The FOB consisted of assessment of gait in an open-field area, grip strength, righting reflex, extensor thrust, response to tail pinch, gross evaluation of auditory response (using a soft clicking stimulus and a loud startle stimulus) and visual and tactile placing. Dams were evaluated on, or shortly after, postpartum Day

Food Consumption: Food consumption was measured for each rat weekly prior to mating, and in the females on gestation Days 0, 4, 7, 14, and 21, and postpartum Days 0 and 4; and in males once a week until sacrifice. Food consumption was not measured during the cohabitation period.
Oestrous cyclicity (parental animals):
Daily vaginal smears, taken from the female rats during the mating period, were evaluted for the presence of sperm and the stage of the estrus cycle.
Sperm parameters (parental animals):
See below in "Postmortem examinations (Parental animals)" for details of sperm parameters and organs.
Litter observations:
Rat pups (F1 generation) were sexed and given a gross external physical examination on postnatal day 0. Dead or stillborn pups were examined for gross external abnormalities. Fresh visceral examinations were not viable due to autolysis.
Postmortem examinations (parental animals):
Clinical Pathology: Five rats/sex/group were fasted overnight for blood collection prior to scheduled necropsy. Blood for clinical chemistry and hematology was collected from the abdominal aorta immediately prior to the scheduled necropsy. For dams, blood was collected from animals in the same or similar physiological state (i.e., postpartum). The following clinical pathology tests were performed:

Clinical Chemistry:

Albumin
A/G ratio
Alanine aminotransferase
Alkaline phosphatase
Aspartate aminotransferase
Bilirubin, Total
Blood Urea Nitrogen
Calcium
Chloride
Cholesterol
Creatinine
Globulin
Glucose
GGT*
Lactate dehydrogenase
Plasma cholinesterase
PO4
Potassium
Protein, Total
Sodium
Triglycerides

*Note: In rats, GGT values are generally 1 or less. When a negative reading is recorded, a value of 0 or <3 is indicated in the raw data. For reporting purposes, all values of <3 were considered to be zero (0) and were reported as such.

Hematology:
Red blood cell count
White blood cell count
Hemoglobin Hematocrit
Mean corpuscular hemoglobin
Mean corpuscular volume
Mean corpuscular haemoglobin concentration
Platelet count
Automated differential Leukocyte count (absolute and relative)
Automated red cell morphology
Reticulocyte count (absolute and relative)

Postmortem Necropsy: Complete necropsies were performed on all parental rats. Rats scheduled for necropsy were fasted overnight and euthanized using sodium pentobarbital. Necropsy included examination of the external surface of the body, all orifices, the cranial, thoracic, and peritoneal cavities, and their contents. Because only two 200 mg/kg/day and no 1000 mg/kg/day treated females gave birth, the uterus of every rat was examined for implants. If implants were visible, the uterus was left intact. If not, a section at the end of the left and right horns was removed, sliced down the middle and put into 10% ammonium sulfide for later examination. Tissues collected at necropsy are listed below (Preserved Tissues). The testes and epididymides were fixed in Bourn's solution; all other tissues were fixed in 10% neutral buffered formalin. Tissues marked with an asterisk (*) below were weighed at scheduled necropsy and organ-to-body weight ratios were calculated.

Preserved Tissues:
Adrenals*
Aorta
Brain (entire)*
Epididymides*
Esophagus
Eyes
Femur with marrow
Harderian gland
Heart*
Intestines (including Peyer's patches)
Cecum
Colon
Duodenum
Ileum
Jejunum
Rectum
Kidneys*
Liver*
Lungs with bronchi
Lymph nodes (mandibular and mesenteric)
Ovaries with oviducts*
Pancreas
Pituitary
Prostate (with seminal vesicles)
Skin (with mammary gland)
Skeletal Muscle and Sciatic Nerve
Spinal Cord (cervical, thoracic. lumbar)
Spleen*
Sternum (bone with marrow)
Stomach
Testes*
Thymus*
Thyroid/parathyroid
Trachea
Urinary Bladder
Uterus (with cervix)
Vagina
Zymbal's gland
Gross lesions
Identification (ear tag)

Histopathologv: The underlined tissues listed above, obtained from 5 rats/sex in the vehicle control and 1000 mg/kg/day groups, were evaluated microscopically by a board-certified veterinary pathologist. The histopathology protocol was amended to include evaluation of the target organs from 5 rats/sex in the 25 and 200 mg/kg/day groups and in recovery males and females. Detailed examination of the testis, epididymis and adrenals were performed, while in females, ovaries, adrenals and liver were examined. The male tissues were processed and stained with PAS/hematoxylin with special emphasis on stages of spermatogenesis and interstitial testicular cell structure. Gross lesions and identified target tissues were evaluated in rats from all dose groups. The tissues were embedded in paraffin, processed by routine histologic methods, and stained with hematoxylin and eosin.
Postmortem examinations (offspring):
Fresh visceral examinations were not viable due to autolysis.
Statistics:
Data were manually collected, entered into Microsoft Excel, and means and standard deviations were calculated using the same software. Data were analyzed for homogeneity of variance using Levene's test. When variances were homogeneous (p>0.001), the data were further analyzed by one-way analysis of variance (ANOVA). When main effect differences were found, all post-hoc comparisons between treated groups were conducted using Dunnett's test. Chi-Square analysis was used for the reproductive performance parameters. Motor activity was analyzed using repeated measures analysis of variance. If a significant trend was observed, data were plotted to determine which, if any, polynomial should be interpreted. In the presence of a significant trend effect, data were then analyzed using analysis of variance followed by a Dunnett's test. Statistical significance was established at p< 0.05. Statistical analyses were performed using Systat 10.
Reproductive indices:
As above "statistics"
Offspring viability indices:
As above "statistics"
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Analytical chemistry
Analyses were conducted on samples of the dosing solutions collected from each preparation. The dosing solutions were homogeneous and proved to be stable for longer than 7 weeks when stored at room temperature. The analytically determined concentrations met the acceptance criteria as specified in the study protocol (i.e. they were within 10% of the target concentrations).

Mortality
None of the animals died during the study.

Clinical observations
In general, no consistent treatment-related clinical signs were noted. Brown discoloration around the mouth was noted in two 1000 mg/kg/day males and females. Other clinical signs were sporadic in nature and included alopecia on the forelimbs and scabbing on the neck. These signs were seen in treated and control groups and were deemed unrelated to treatment. Recovery animals treated with 100 mg/kg/day exhibited similar clinical signs (scab/sore on neck, discoloration around mouth/nose, alopecia). One animal had an injured hind-limb, which was not related to treatment.

Food consumption
In general, limited treatment-related effects were noted on food consumption. During week 1, the 1000 mg/kg/day treated males had significantly decreased food consumption compared to controls. A similar trend was seen in the 1000 mg/kg/day males designated for recovery. During the later part of gestation, dam food consumption was lower in the 200 mg/kg/day and 1000 mg/kg/day treated groups; this was attributed to the reduced number of gravid dams in these groups. Satistically significant increases in food consumption were noted but these were considered to be coincidental and unrelated to treatment.

Body weights and body weight gain
Overall, treatment with Phosflex TXP had limited effects on body weight and body weight gain. However, body weights/body weight gains were reduced in males (core and recovery) treated with 1000 mg/kg/day after 1 week of treatment. This change correlated well with the reduced food consumption seen during the same time. No other biologically relevant differences were found in the body weights/body weight gain of males. During the later part of gestation, dam body weights/body weight gains were lower in the 200 and 1000 mg/kg/day treated groups; this was attributed to the lower number of gravid dams in these groups. Conversely, gestation body weight/body weight gain for recovery dams previously treated with 1000 mg/kg/day were increased compared to recovery vehicle controls. Statistically significant increases in body weight/body weight gain were noted sporadically during the study. Pre-terminal and fasted weights of females treated with 1000 mg/kg/day were significantly increade compared to controls. Recovery males treated with 1000 mg/kg/day had increased body weight gain during recovery week 2 and 7; this resulted in an increased total gain for this group. In recovery females, body weight gain in the 1000 mg/kg/day group was increased during week 3 and decreased during recovery week 1.

Reproductive performance
After mating, all but one female in the 1000 mg/kg/day treated group were sperm-positive. Thus, no differences in percent mated were noted between the treated and control groups. However, the number of dams that successfully underwent parturition (successful mating outcome) was significantly decreased in the 200 and 1000 mg/kg/day treated groups (2 out of 11 and 0 out of 10 in the 200 mg/kg/day and 1000 mg/kg/day treated groups, respectively). The percent successful parturition translated into 100% in the 25 mg/kg/day group, 18% in the 200 mg/kg/day group and 0% in the 1000 mg/kg/day group, compared to 100% in the vehicle control group. The number of gravid dams was similar to the number undergoing parturition ; staining of the uterus revealed only 1 additional 1000 mg/kg/day treated animal as pregnant; as such, the lower pregnancy rate did not result from post-implantation loss. Of the dams that underwent successful parturition, the average length of gestation was unaffected by treatment and was approximately 22 days for each group. The pronounced reproductive effects seen in the 200 and 1000 mg/kg/day treated groups led to mating of the recovery animals. Recovery animals were mated within group (i.e. recovery control males were mated with control females, while recovery high dose males were mated with high dose recovery females). A cross-over mating (high dose males with naive females) was performed first in an effort to determine if the observed effects were male or female mediated. Recovery males were held for four weeks after dosing cessation prior to conducting the cross-over mating, while recovery females were held for three weeks prior to conducting the within group mating. The cross-over mating reulted in 5 pregnant (naive) dams, showing that the high dose males were fertile after only 4 weeks of recovery. For the within group matings, 60% of the recovery control dams and 100% of the 1000 mg/kg/day recovery dams were sperm positive. Successful parturition was seen for all sperm positive dams, resulting in 100% pregnancy in the recovery groups (controls and high dose). The average length of gestation was similar among the recovery dams. These findings suggest full recovery from the observed functional deficit in reproductive performance.

Clinical chemistry
Statistically significant differences were noted in various clinical chemistry parameters between treated and control groups. Specifically, ALKP was decreased in the 200 and 1000 mg/kg/day males , while BUN, ALT and calcium were increased. Also, LDH values were significantly decreased in the 1000 mg/kg/day males. In the females, the ALKP showed a statistically significant, dose related decline in all treated groups, which may have been related to treatment, but the clinical significance of this is unclear. In addition, cholesterol and phosphate were significantly increased, while A/G ratios were significantly decreased in the 1000 mg/kg/day group. ALT levels were increased in the 200 and 100 mg/kg/day treated females, but only significantly so in the 200 mg/kg/day group. In the recovery groups, the 1000 mg/kg/day males had significantly increased calcium and phosphate levels. No other changes were noted in the clinical chemistry parameters.

Hematology
Statistically significant decreases were noted in hemoglobin, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in the 1000 mg/kg/day females. The changes noted were minor in nature and fell within historical control values for non-pregnant Sprague-Dawley rats. In core males, percent eosinophils were decreased in the 200 and 1000 mg/kg/day groups, while core females treated with 1000 mg/kg/day had significantly increased absolute neutrophils. Also percent LUC (large unstained cells) was significantly increased in the 1000 mg/kg/day recovery males. Recovery females treated with 1000 mg/kg/day had reduced basophils. No difference in the relative or absolute reticulocyte counts were found among the groups.

Plasma cholinesterase
Plasma cholinesterase (PChE) values were significantly decreased in the 200 and 1000 mg/kg/ day males and females compared to controls. Cessation of treatment resulted in reversal of this effect as no differences were noted in PChE following the recovery period. It should be noted that values of both the control and 1000 mg/kg/day recovery males were lower than other reported values in this study for no apparent reason; these samples were rerun to confirm the lower values seen during recovery.

Functional observation battery and motor activity
No differences were noted in the FOB parameters among the core groups. However, in the recovery groups, the 1000 mg/kg/day treated females had significantly decreased forelimb grip strength compared to controls. This change was considered incidental in nature since this finding was not noted in the core animals. No significant differences were found in the motor activity parameters of any group in this study.

Organ weights
Statistically significant differences were found in several organ weight parameters. Absolute heart weight and heart to brain weight ratio were significantly decreased in the 1000 mg/kg/day treated rats (both sexes). Absolute adrenal weights and adrenal weight ratios were elevated in 200 and 1000 mg/kg/day treated rats (males and females); an increase was also noted in the 25 mg/kg/day females. Absolute liver weights and liver weight ratios were increased in both sexes of rat treated with 1000 mg/kg/day; this change was also noted in males treated with 200 mg/kg /day. Reproductive organ weights were affected by treatment; for males, absolute testes and epididymides weights and their ratios were significantly reduced in the 1000 mg/kg/day treated group. For the females, ovary weights and ratios were significantly elevated in the 200 and 1000 mg/kg/day treated groups. In addition, brain to body weight ratios were increased in the 1000 mg/kg/day treated females. After the recovery period, organ weight changes resolved for the reproductive organs, but persisted in some of the other organs. Changes were still present in adrenal to brain weight ratios of males, liver of females and in brain to body weight and heart to brain weight ratios in both sexes. Histopathological lesions were noted in the adrenals, liver (females only), testes and ovaries of treated animals, which correlate with the organ weight changes described above.

Gross pathology
Gross necropsy findings were limited, generally singular in occurrance, and seen across all groups including controls; therefore they were deemed unrelated to treatment. Additionally, the uterus of non-gravid dams were stained with 10 % ammonium sulfide in an effort to determine if implantation had occurred. Only one dam treated with 1000 mg/kg/day was identified as being gravid based on the results of staining.

Histopathology
Lesions consisted of degeneration of the germinal epithelium of the testeswith corollary findings of sloughed epithelial cells in the lumen of the epididymis (all three dose levels). Findings in the ovaries consisted of distinct mild diffuse hyperplasia of the interstitial cells (all three dose levels). Diffuse cytoplasmic vacuolation was noted in the adrenals (all dose levels of males and in the 200 and 1000 mg/kg/day treated females); minimal to mild fatty vacuolation of individual hepatocytes was seen in the 200 and 1000 mg/kg/day females. Overall, the incidence and severity of the histopathological changes were less pronounced at the lower doses (25 and 200 mg/kg/day). Recovery males were euthanized and necropsied at the end of the mating trials (after 7.5 weeks) and recovery females were necropsied after postnatal day 4 (following 9 weeks of recovery). Following the recovery period, the incidence and severity of all changes were decreased in the target tissues of the 1000 mg/kg/day group, indicating ongoing, sucessful repair.
Key result
Dose descriptor:
NOAEL
Effect level:
< 25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
25 mg/kg bw/day (nominal)
System:
other: haematopoietic, male/female reproductive systems/haematopietic/autonomic nervous system
Organ:
other: adrenals, testes, epididymides, ovaries, heart and liver were identified as target organs
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Litter survival and pup body weights
Evaluations of litter survival and offspring body weights were limited to those dams that underwent successful parturition. Staining of the uterus revealed only one additional high-dose dam as pregnant. As such, post-implantation loss was not a cause for the reduction in pregnancy noted in the 200 and 1000 mg/kg/day groups. No statistically significant differences in mean litter size or the number of stillborn fetuses were detected among the groups. Neither the core nor the recovery dams showed differences in litter size compared to the vehicle controls. While the percentage of pups surviving to Day 4 and the litter sizes were not different among the core group females, it is important to note that only 2 females in the 200 mg/kg/day treated group underwent parturition. Offspring body weights and growth were similar across all groups and unaffected by treatment. Coincidentally, the cross-over naive dams had significantly heavier offspring on postnatal day 0 compared to vehicle controls. This trend continued, but by postnatal day 4, the offspring body weights were no longer significantly elevated.
Key result
Remarks on result:
other: Not specified
Key result
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Oral administration of Phosflex TXP to Sprague-Dawley rats at doses of 25, 200 and 1000 mg/kg/day did not result in any overt signs of parental toxicity or relevant changes in food consumption, body weight, body weight gain, or motor activity. Reproductive outcome (successful pregnancy) was adversely impacted in the 200 and 1000 mg/kg/day treated groups. Mating of the recovery animals showed complete reversal of the effects seen in reproductive performance. Since reversal was seen in both sexes, it was not possible to determine if the effect was male- or female- mediated. Clinical pathology profiles revealed relevant changes in serum chemistry (blood urea nitrogen and ALT were elevated in one or both sexes of the 200 or 1000 mg/kg/day groups, while plasma cholinesterase activity was reduced across both sexes of the 200 or 1000 mg/kg/day groups). Based on organ weight data, the adrenals, testes, epididymides, ovaries, heart and liver were identified as target organs. With the exception of the heart, treatment-related histological lesions were observed in these organs [adrenals, testes, epididymides, ovaries, liver (females only)]. Following the recovery period, the incidence and severity of all histological changes were decreased in the target tissues of 1000 mg/kg/day group, indicating ongoing, successful repair.
Based on the findings of this study, the no-observed-adverse-effect-level (NOAEL) of Phosflex TXP was less than 25 mg/kg.
Executive summary:

Phosflex TXP was administered in a vehicle of corn oil by oral gavage at dose levels of 0 (vehicle control), 25, 200 or 1000 mg/kg/day to 11 Sprague-Dawley rats/sex/group for the assessment of reproductive and developmental toxicity, and target organ toxicity. Rats were dosed 7 days/week for 2 weeks prior to mating, during the 2-week mating period, and throughout gestation and lactation until sacrifice, for a total of approximately 33 days of dosing for males and 48 days for females. Doses were administered at a constant volume of 2 ml/kg. The control and high dose groups included an additional 5 rats/sex designated as recovery animals. During the recovery phase of the study, the protocol was amended to include a mating of the recovery animals as well as a cross-over mating. The cross-over mating consisted of cohabitation of high dose males with naive females, following which recovery animals were mated within their respective groups. The mating of recovery animals was added to the study in order to determine whether the apparent adverse effects of test material on reproductive function were reversible, whereas cross-over mating was used to aid in determining whether the observed effects were male- or female-mediated. Recovery males were held for 4 weeks after dosing cessation prior to conducting the cross-over mating, while recovery females were held for 3 weeks prior to performing the within-group mating. Recovery males were euthanized and necropsied at the end of the mating trials (after llA weeks) and recovery females were necropsied after postnatal day 4 (9 weeks of recovery). Parental food consumption, body weight, body weight gain, reproductive performance, organ weight and histopathology were evaluated during the study, along with fetal body weights and survival. Food consumption, body weight and body weight gain were slightly reduced in the 1000 mg/kg/day group after the first week of treatment. During the later part of gestation, dam food consumption, body weight and body weight gain were lower in the 200 and 1000 mg/kg/day groups; this was attributed to the lower rate of successful pregnancies. The number of successful matings (sperm positive) was similar across all groups (11/11 mated in the control, 25 and 200 mg/kg/day groups, while 10/11 of the 1000 mg/kg/day group rats were sperm-positive). However, reproductive outcome was adversely impacted in the 200 and 1000 mg/kg groups. Successful parturition was 100% in the control and 25 mg/kg/day groups, and 18% and 0% in the 200 and 1000 mg/kg/day groups, respectively. Litter size, survival and offspring body weight were unaffected by treatment at 25 mg/kg/day. The pronounced reproductive effects seen in the 200 and 1000 mg/kg/day groups led to mating of the recovery animals. The cross-over mating (naive females x high dose males) was performed first and resulted in all 5 naive females becoming pregnant, showing that the high dose males were fertile after only 4 weeks of recovery. For the within-group matings, 60% of the recovery control dams and 100% of the 1000 mg/kg/day recovery dams were sperm-positive. Successful parturition was seen for all sperm-positive dams resulting in 100% pregnancy in the recovery groups (controls and high dose). These findings suggest resolution of the observed functional deficit in reproductive performance. Thus, the functional deficit in reproductive performance was reversed in males after 4 weeks of recovery and after 3 weeks for females. Since reversal was seen in both sexes, it was not possible to determine if the effect on reproductive performance was male- or female-mediated. Clinical pathology profiles revealed relevant changes in serum chemistry (blood urea nitrogen, and alanine aminotransferase were elevated in one or both sexes of the 200 or 1000 mg/kg/day groups, while plasma cholinesterase activity was reduced across both sexes of the 200 or 1000 mg/kg/day groups). Other clinical chemistry changes were noted, but the biological significance is unclear, since the changes were either subtle in nature (Ca) or the direction of change (decreased alkaline phosphatase) has no clinical significance. No differences in functional observation battery (FOB) or motor activity parameters were noted in the core groups. Gross necropsy findings were limited, generally singular in nature and seen across all groups, including controls, therefore, they were deemed unrelated to treatment. Based on organ weight data, the adrenals, testes, epididymides, ovaries, heart and liver were identified as target organs. With the exception of the heart, treatment-related histological findings were observed in these organs [adrenals, testes, epididymides, ovaries, liver (females only)]. Lesions consisted of degeneration of the germinal epithelium of the testes with corollary findings of sloughed epithelial cells in the lumen of the epididymis (all three dose levels). Findings in the ovaries consisted of distinct mild diffuse hyperplasia of the interstitial cells (all three dose levels). Diffuse cytoplasmic vacuolation was noted in the adrenals (all dose levels of males and in the 200 and 1000 mg/kg/day treated females); minimal to mild fatty vacuolation of individual hepatocytes was seen in the 200 and 1000 mg/kg/day females. Overall, the incidence and severity of the histopathological changes were less pronounced at the lower doses (25 and 200 mg/kg/day). Following the recovery period, the incidence and severity of all changes were decreased in the target tissues of 1000 mg/kg/day group, indicating ongoing, successful repair. Based on the findings of this study, the no-observed-adverse-effect-level (NOAEL) of Phosflex TXP for was less than 25 mg/kg/day. On the basis of the results presented within the data, the results were determined to triggered classification under the Dangerous Substance Directive (67/548/EEC) or the CLP Regulation (EC No 1272/2008) as follows:

Repr. Cat. 3; R62 Possible risk of impaired fertility

Repro Cat 2; H361: Suspected of damaging fertility or the unborn child (testes, epididymides, ovaries)

Subsequent to this it has been noted that in COMMISSION REGULATION (EU) No 618/2012 of 10 July 2012 amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures, the reproduction toxicity of this substance has subsequent been harmonised . As a result of this, and to bring the registration dossier in accordance with this regulation, the classification and labelling of the substance has been adjusted as follows in accordance with CLP Regulation (EC No 1272/2008) index no015-201-00-9 as follows:

 

Repr. Cat. 2;R60: May impair fertility

 

Repr. Cat 1b, H360f: May damage fertility or the unborn child(Testicular effects; oral)

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
62.5 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
k2
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In the main screening study, the substance was administered in a vehicle of corn oil by oral gavage at dose levels of 0 (vehicle control), 25, 200 or 1000 mg/kg/day to 11 Sprague-Dawley rats/sex/group for the assessment of reproductive and developmental toxicity, and target organ toxicity.

Reproductive outcome was adversely impacted in the 200 and 1000 mg/kg groups. Successful parturition was 100% in the control and 25 mg/kg/day groups, and 18% and 0% in the 200 and 1000 mg/kg/day groups, respectively. Litter size, survival and offspring body weight were unaffected by treatment at 25 mg/kg/day. The pronounced reproductive effects seen in the 200 and 1000 mg/kg/day groups led to mating of the recovery animals. The cross-over mating (naive females x high dose males) was performed first and resulted in all 5 naive females becoming pregnant, showing that the high dose males were fertile after only 4 weeks of recovery. For the within-group matings, 60% of the recovery control dams and 100% of the 1000 mg/kg/day recovery dams were sperm-positive. Successful parturition was seen for all sperm-positive dams resulting in 100% pregnancy in the recovery groups (controls and high dose). These findings suggest resolution of the observed functional deficit in reproductive performance. Thus, the functional deficit in reproductive performance was reversed in males after 4 weeks of recovery and after 3 weeks for females. Since reversal was seen in both sexes, it was not possible to determine if the effect on reproductive performance was male- or female-mediated. Based on organ weight data, the adrenals, testes, epididymides, ovaries, heart and liver were identified as target organs. With the exception of the heart, treatment-related histological findings were observed in these organs [adrenals, testes, epididymides, ovaries, liver (females only)]. Lesions consisted of degeneration of the germinal epithelium of the testes with corollary findings of sloughed epithelial cells in the lumen of the epididymis (all three dose levels). Findings in the ovaries consisted of distinct mild diffuse hyperplasia of the interstitial cells (all three dose levels). Overall, the incidence and severity of the histopathological changes were less pronounced at the lower doses (25 and 200 mg/kg/day). Following the recovery period, the incidence and severity of all changes were decreased in the target tissues of 1000 mg/kg/day group, indicating ongoing, successful repair. Based on the findings of this study, the no-observed-adverse-effect-level (NOAEL) of the was less than 25 mg/kg/day.

A read across studies to the analogue Tris(methylphenyl) phosphate, CAS 1330-78-5 which is considered an appropriate structural analogue is also presented.  Effects were noted at all dose levels in the study utilised for the assessment; in the main, these were related to male reproductive effects and included such effects as sperm concentration and morphology and sperm motility being decreased in the dose groups tested. As effects were noted at all dose levels, it was not possible to determine a NOEC for this this endpoint on the basis of the studies included. However, no further studies on this endpoint are proposed on the grounds that other effects (specifically neurotoxicity) are observed at lower dose ranges. As hazard prevention is required for this endpoint, it is considered that reproductive effects could be considered as secondary and hence be addressed by risk mitigation factors.

Short description of key information:

Reproduction effects are discussed below, including those on the analogue Tris(methylphenyl) phosphate, CAS 1330-78-5

Justification for selection of Effect on fertility via oral route:

GLP, carried out according to recognised guideline.  Also see discussion below

Effects on developmental toxicity

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
in second species.
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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.
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
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.
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
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.
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
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
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.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
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.
Key result
Dose descriptor:
other: Not determined
Remarks on result:
not measured/tested
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

 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

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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Read across to CAS (68937 -41 -7) Effect on developmental toxicity: via oral route:

A NOAEL of 200 mg/kg/day is documented for this endpoint on the basis of the maternal toxicity observed.

 

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.

 

All control and treated animals survived to scheduled termination. Salivation was observed in each of the test substance-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 the test substance 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 the test substance, 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. The test substance was not teratogenic in the rat at the dose levels tested.

Toxicity to reproduction: other studies

Additional information

See below

Justification for classification or non-classification

In 2004 a study was carried out to provide information on possible health hazards likely to arise from repeated exposure to trixylyl phosphate (TXP), over a relatively limited period of time - the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test [OECD 422].

The results of this study led to the conclusion that TXP had the potential to cause adverse effects on reproduction. Effects on fertility, specifically a dose-related effect on parturition (100% success at 25 mg/Kg/day; 18% at 200 mg/Kg/day; 0% at 1000 mg/Kg/day) was observed in rats exposed to TXP via the oral route.

As a direct consequence of this screening study, Industry (specifically the manufacturer Supresta) voluntarily classified and labelled the chemical as a Category 3 reproductive hazard for fertility – R62 in accordance with the Guidance issued in Annex VI to Directive 67/548/EEC and its amendments. A dossier was submitted to the Netherlands authorities as is required under EC legislation since TXP was not officially classified or labelled in Annex I of Directive 67/548/EEC at the time.

 

In October 2008 RIVM, the Netherlands drafted an Annex XV Dossier containing a Proposal for a Harmonised Classification and Labelling of TXP. The proposal suggested Toxic for Reproduction, Category 2 R60. The registrant disagreed with this classification and labelling and communicated the proposed classification and labelling in the lead registration dossier submitted in accordance with the 2010 deadline.

 

Subsequent to this it has been noted that in COMMISSION REGULATION (EU) No 618/2012 of 10 July 2012 amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures, the reproduction toxicity of this substance has subsequent been harmonised . As a result of this, and to bring the registration dossier in accordance with this regulation, the classification and labelling of the substance has been adjusted as follows in accordance with CLP Regulation (EC No 1272/2008) index no 015-201-00-9 as follows:

 

Repr. Cat 1b, H360f: May damage fertility or the unborn child(Testicular effects; oral)