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Toxicological information

Toxicity to reproduction

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Administrative data

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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phosflex TXP
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: not specified
- Physical state: liquid
- Analytical purity: not specified
- Impurities (identity and concentrations): not specified
- Composition of test material, percentage of components: not specified
- Isomers composition: not specified
- Purity test date: not specified
- Lot/batch No.: 02223D0200 T#127
- Expiration date of the lot/batch: not specified
- Radiochemical purity (if radiolabelling): not applicable
- Specific activity (if radiolabelling): not applicable
- Locations of the label (if radiolabelling): not applicable
- Expiration date of radiochemical substance (if radiolabelling): not applicable
- Stability under test conditions: stability of the test material in the vehicle (i.e. corn oil) was established by Experimur during a 14-day range-finding study.
- Storage condition of test material: room temperature, protected from light.
- Other:

Test animals

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).

Administration / exposure

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:
Doses / concentrationsopen allclose all
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

Examinations

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"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

Key result
Remarks on result:
other: Not specified

Target system / organ toxicity (F1)

Key result
Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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)