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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 19 2004 to March 21 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, carried out according to recognised guideline

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
minor deviations in timing of 3 observations; animal room humidity exceeded the limits specified in the guideline by 3% for 1 day; in the opinion of the Study Director, these minor deviations did not affect the quality or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Kronitex TCP
IUPAC Name:
Kronitex TCP
Details on test material:
- Name of test material (as cited in study report): Kronitex TCP
- 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: Clear liquid
- Analytical purity: Not specified
- Impurities (identity and concentrations): 0.01% water
- Composition of test material, percentage of components: not specified
- Isomers composition: not specified
- Purity test date: October 6th 2003
- Lot/batch No.: 20015254
- Expiration date of the lot/batch: Assume stable for duration of study
- 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: not specified
- Storage condition of test material: room temperature.
- Other:

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
On February 3 (41 males) and February 10 (165 females), 2004, Sprague-Dawley (Crl: CD (SD)IGS BR) rats were received from Charles River Laboratories, Raleigh, North Carolina. All female rats were approximately nine weeks old at arrival, and ten weeks old at breeding. The male rats, utilized for mating purposes only, were approximately ten weeks old at arrival and 12 weeks old at breeding. Only females with positive evidence of mating (vaginal plug or sperm) were selected for study. These animals were weighed on Day 0 of gestation which was defined as the day when evidence of mating was observed. The females weighed between 187 to 267 g on Day 0 of gestation. During the nine to 16-day acclimation period, all rats were observed daily for any clinical signs of disease.
Mated female rats were given a clinical examination on Day 0 of gestation and only females considered suitable, based on the results of these examinations, were included in the selection process. Animals were sorted into treatment groups using a simple randomization procedure on the day they mated. After the required numbers of mated females were sorted among the groups, the males were euthanized via carbon dioxide inhalation and discarded. Extra female rats obtained but not used on study, were either euthanized via carbon dioxide inhalation and discarded or transferred to the stock colony.
Each female rat was assigned an animal number to be used in Provantis™ and implanted with a microchip bearing a unique identification number. Each cage was identified by the study number, animal number, group number, and sex. The individual animal number plus the study number comprised a unique identification for each rat. Animal identification was verified during the conduct of the study, as documented in the study data.
From acclimation until euthanasia, the rats were individually housed in suspended, stainless steel, wire-mesh type cages, except during pairing when the females were housed in similar cages overnight with males (1 :1). Fluorescent lighting was provided for approximately 12 hours per day via automatic timer. Throughout the study, all rats were kept in an environmentally controlled room. Temperature and relative humidity in the animal room were monitored and recorded daily, and maintained between 64 to 77°F and 32 to 73%, respectively.
Diet (meal lab Diet Certified rodent diet #5002, PMI Nutrition International, Inc.) and tap water were available ad libitum during the course of the study. Documentation of lot numbers of the basal diet used is retained in the study file. Analytical certifications of each diet lot were performed by the manufacturer and are maintained in the Archives. Water was supplied using an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. 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 this study.

Mated female rats were given a clinical examination on Day 0 of gestation and only females considered suitable, based on the results of these examinations, were included in the selection process. Animals were sorted into treatment groups using a simple randomization procodure on the day they mated. After the required numbers of mated females were sorted among the groups, the males were euthanized via carbon dioxide inhalation and discarded. Extra female rats obtained but not used on study, were either euthanized via carbon dioxide inhalation and discarded or transferred to the stock colony.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
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, or in consultation with the Sponsor, on the basis of available date from previous studies.

Vehicle and Test Article Preparation: To prepare the vehicle, the required amount of corn oil was dispensed into amber glass containers prior to handling the test article. The vehicle was dispensed weekly and stored refrigerated. To prepare the test article formulations, the required amount of test article, TCP, was weighed directly into a beaker. Vehicle was added to the beaker and the contents were stirred using a magnetic stir bar and stir plate until well mixed. The mixture was transferred into a graduated cylinder. The beaker was rinsed with additional vehicle and the rinse was transferred into the cylinder. More vehicle/was added to the cylinder to yield the required amount of prepared test article. The cylinder was shaken thoroughly and the mixture was dispensed into a beaker. The contents of the beaker were stirred using a magnetic stir bar and stir plate, and dispensed into amber glass containers using a syringe. The test article formulations were prepared weekly and stored refrigerated. Each formulation was prepared separately. The test article was used as received from the Sponsor and no adjustment was made for purity.

Administration: Test article and vehicle control administration began on Gestation Day 0 and continued through to include Day 19. The test article was administered to the treated groups via oral gavage once per day at approximately the same time each day at respective dose levels of 20, 100, 400, and 750 mg/kg/day at a dose volume of 5 mL/kg/dose. The control animals received the vehicle, com oil, at the same volume, duration, and dosing regimen as the treated animals. Individual doses were based on the most recent body weight. The vehicle and test article were administered via oral gavage using an appropriately sized plastic disposable syringe attached to a Fuchigami dosing needle. The test article formulations were stirred continuously during test article administration using a magnetic stir bar and stir plate.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dosing Formulations
Homogeneity: Homogeneity of the test article in the vehicle at similar concentrations and batch sizes used in this study was established in a previously conducted study (MPI Research Study Number 1038-002).
Stabilty: Refrigerated stability of the test article formulations for at least 14 days at concentration levels of 10 to 200 mg/mL was established in a previously conducted study (MPI Research Study Number 1038-001). Samples (5 mL/sample) of the low concentration formulation (4.0 mg/mL) from Week 1 were collected from the container with a syringe and placed in an amber glass bottle. The samples were stored refrigerated for up to 14 days, and delivered to MPI Analytical after 7 and 14 days for analysis of the stability of the test article in the vehicle.
Concentration: Samples (5 mL/sample) of the test article formulations at each concentration were collected from the preparations for Weeks 1 and 4 of study. The samples were collected from the container, while stirring, with a syringe, placed into amber glass bottles, and stored refrigerated until analyzcd for test article concentrations. Additional samples (2 mL/sample) of the test article formulations at each concentration were collected from Week 3 preparations and retained frozen for possible further analysis.
Analyses: All analytical work was conducted by MPI Analytical, a division of MPI Research, using the methodology from an analytical method validated by KAR Laboratories, Kalamazoo, Michigan. MPI Analytical conducted a partial (transfer) validation in accordance with the appropriate SOPs, using validated procedures from KAR Laboratories. All samples were analyzed using the procedures validated by MPI Analytical.
Reserve Sample and Test Article Disposition: a reserve sample from the lot ofTCP used in this study was taken and archived. The remaining test article will be returned to a Sponsor-designated location after completion of the study.

A High-Performance Liquid Chromatography - Ultraviolet (HPLC-UV) assay has been validated for the test article, tricresyl phosphate (TCP), in corn oil for concentrations ranging from 2.5 to 200 mg/mL. The validation procedure consisted of four separate validation runs occurring over separate days. The first run consisted of six injections of a working RF standard, a check standard, a diluent blank, a matrix blank, and system performance checks. The first run included triplicate dilutions taken from each of three Quality Control (Qc) dose preparations at approximately 2.5 and 20 mg/mL. The second run included triplicate dilutions taken from each of three QC dose preparations at approximately 2.5, 20, and 200 mg/mL. The third run included re-prepared triplicate dilutions of the 200 mg/mL QC dose concentration. Autosampler stability samples and samples for determining the stability under the conditions of administration were analyzed during the second, third, and fourth analytical runs. The selectivity, linearity, range, accuracy, and precision of the method were evaluated. Linearity around working response factor (RF) was determined at +/- 30%. All coefficients of
determination (R2) values were greater than 0.99. The calculated concentrations of the response factor standards over the four runs ranged from - 1.2 to 1.0% RE. Analysis of the matrix was performed over three validation runs and analysis of the diluents blank was performed over all validation runs. None of the matrix blank or diluent assays contained any interferences within the retention region ofTCP, indicating that the method was selective for quantitation. The recoveries of TCP in the QC preparations ranged from 97.7 to 101.6% at 2.5 mg/mL, 96.4 to 103.2% at 20 mg/mL, and 94.1 to 102.3 % at 200 mg/mL, over the three validation runs. The intra-day accuracy (%RE) of the QC samples ranged -0.1 to 1.6% at 2.5 mg/mL and -0.1 to 3.2% at 20 mg/mL for the first validation run and ranged -2.3 to -1.0% at 2.5 mg/mL, -3.6 to -3.3% at 20 mg/mL, and -5.9 to -3.1 % at 200 mg/mL for the second validation run, and -2.4 to 2.3% at 200 mg/mL for the third validation run. The intra-day precision (%RSD) of the QC samples were 0.9 and 0.7% RSD at 2.5 mg/mL, 1.7 and 0.1 % RSD at 20 mg/mL, and 2.6 and 1.5% at 200 mg/mL, for the validation runs. Pre-processed QC samples were stable at ambient temperature over the 6 days tested. The post-processed samples and selected standards were stable at ambient temperature over the 7 days tested.
Details on mating procedure:
Female rats were housed together with an untreated male rat (1 :1i) used specifically for breeding. Males were of the same strain and from the same source as the females. Mating was established by daily inspection for a copulatory plug in the vagina or microscopic observation of sperm in the vaginal rinse. Evaluations for evidence of mating was performed early each morning before 9:00 AM. The day evidence of mating was confirmed wil be designated Day 0 of gestation. Once all mated females have been identified each day they weresorted into appropriate groups.
Duration of treatment / exposure:
Test article and vehicle control administration began on Gestation Day 0 and continued through to include Day 19.
Frequency of treatment:
The test article was administered to the treated groups via oral gavage once per day at approximately the same time each day.
Duration of test:
28 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
20 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
750 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Number of animals
Group Dose level mg/kg/day Initial Laparohysterectomy/necroscopy Microscopic pathology
1 0 25 25 As required
2 20 25 25 As required
3 100 25 25 As required
4 400 25 25 As required
5 750 25 25 As required

Examinations

Maternal examinations:
In-life Examinations
Mortality and Cageside Observations: All rats were observed twice each day for morbidity, mortality, signs of injury, and availability of food and water.
Detailed Clinical Examinations: Detailed clinical examinations were conducted daily from Gestation Days 0 through 20. Each rat was removed from the cage and given a detailed clinical examination, approximately one hour postdose. Any pretest observations and observations conducted on the male rats used for breeding purposes are not reported, but are maintained in the study file.

Body Weights and Body Weight Changes: Individual body weights were recorded on Gestation Days 0, 3,6, 9, 12, 15, 18, and 20. Individual body weight change was calculated for the following gestation day intervals: 0-3, 3-6, 6-9, 9- 1 2, 12-15, 15-18, 18-20, and 0-20. Adjusted body weight (Day 20 gestation body weight minus the gravid uterine weight) and adjusted body weight change (Days 0-20 of gestation) were also calculated. Any pretest body weights and body weights recorded for the male rats used for breeding purposes are not reported, but are maintained in the study file.
Food Consumption: Food consumption was recorded on the corresponding body weight days and calculated for the following intervals: Gestation Days 0-3, 3-6,6-9,9-12, 12-15, 15-18, 18-20, and 0-20.

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. Gross lesions were saved in 10% neutral buffered formalin, and the carcasses were discarded.

Ovaries and uterine content:
Ovarian and Uterine Examinations: On Day 20 of gestation, each female was euthanized by carbon dioxide inhalation and immediately subjected to a laparohysterectomy. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous abnormalities. The abdominal cavity was then opened and the uterus was exposed. The uterus was excised and the gravid uterine weight 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, position of the cervix, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded. 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 grossly examined. Before the umbilical cord was cut
on each fetus, it was momentarly clamped with forceps to prevent bleeding and promote clotting. After examination, the uterus was discarded. Each implant was characterized as either a viable or nonviable fetus, or either an early or late resorption. Viable fetuses responded to touch while nonviable fetuses did not and showed no signs of autolysis. Early resorptions were characterized as implantation sites consisting of tissues but no recognizable fetal characteristics, while late resorptions displayed recognizable fetal characteristics, but were undergoing the process of autolysis. Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites. If no foci were seen, the female was considered not pregnant and all data were excluded from statistical analysis.
Fetal examinations:
Teratologic Examinations: Fetuses were individually weighed, sexed externally, tagged for identification, and examined for external malformations and variations. Approximately one-half of the fetuses in each litter were placed in Bouin's solution for subsequent soft tissue examination using the Wilson razor-blade sectioning technique. The remaining fetuses were fixed in alcohol, processed for Alizarin Red Sand Alcian blue staining, and cleared with glycerin for subsequent skeletal examination of bone and cartilage. Fetal findings were classified as malformations or developmental variations under the supervision of a developmental toxicologist.
Statistics:
The statistical analyses employed for various endpoints are listed below. (Control group = 1, treatment groups = 2,3,4,5)
Parental in-life data
Endpoints: gestation body weights, gestation body weight increases, gestation food consumption, adjusted body weights, adjusted body weight changes - statistical test: group pair-wise comparisons.
Fertility Indices
Endpoint: Pregnancy index - statistical test: Fisher's exact test
Uterine and Ovarian exam
Endpoints: Gravid uterine weights, Corpora lutea/dam, total implantations/dam, Litter size (per dam), viable fetuses/dam, total number resorptions/dam, number early resorptions/dam, number late resorptions/dam - statistical test: group pair-wise comparisons.
Endpoints: fetal sex ratio (% males/litter), % preimplantation loss (mean/dam), % postimplantation loss (mean/dam) - statistical test: arcsin-square root transformation.
Endpoints: malformations by finding and exam type (external, visceral and skeletal)-litter incidence, variations by finding and exam type (external, visceral and skeletal)-litter incidence, total malformations by finding and exam type (external, visceral and skeletal combined)-litter incidence - statistical test: Fisher's exact test (Fetal and litter incidences were reported but only the litter incidences were statistically analysed).
Endpoint: non-viable fetuses per dam - statistical test: descriptive statistics.
Endpoint: mean fetal body weights - statistical test: covariate analysis.

Indices:
Mean Fetal Body Weights

The mean fetal body weights and litter sizes were collected from each dam. As outlined in the protocol, the analysis for fetal bodyweights included tests to determine if it was appropriate to conduct an analysis of covariance using litter size as a covariate. If the assumptions for the covariate failed, the model was run with just treatment as an effect and a follow-up group pair-wise analysis was run. If the assumptions did not fail, the model was run with treatment and litter size and pairwise comparisons were made using Dunnett's test.

Upon examination of the data from the above analysis, a follow-up trend analysis was requested.

A dose-response analysis (linear trend test) was conducted using linear contrasts under the appropriate statistical modeL. The dose-response analysis was run for males, females and pooled sexes.

Males
The assumptions on the analysis of covariance were violated and thus, pair-wise comparisons were performed without the covariate. Treatments 2, 4 and 5 were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattem in the data across treatments (p = -(0.0001).

Females
The assumptions on analysis of covariance were met and thus, an analysis of covariance was performed with the litter size as the covariate. Pair-wise comparisons showed that all treatments were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattern in the data across treatments (p = -(0.0001).
Historical control data:
Yes; documented in Appendix L of the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day, the only maternal toxicity seen was an increased frequency of salivation following dosing. At the higher dose levels evaluated (400 and 750 mg/kg/day), the frequency of animals with sparsc amounts of hair in the abdominal and lumbar regions, ventral surface, and hind limbs, and unkempt appearance was also increased. Lower body weights and body weight gain during gestation were seen at 400 and 750 mg/kg/day and reduced food consumption was seen at 750 mg/kg/day. Excessive feed spilage was also seen with increased frequency among females in the 400 and 750 mg/kg/day groups. No effect of treatment was evident from maternal macroscopic findings, uterine implantation data, fetal sex ratios, or fetal malformation data.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
See below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
See below
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
See below
Urinalysis findings:
no effects observed
Description (incidence and severity):
See below
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See below
Gross pathological findings:
no effects observed
Description (incidence and severity):
See below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
See below
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
See below
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
See below
Early or late resorptions:
no effects observed
Description (incidence and severity):
See below
Dead fetuses:
no effects observed
Description (incidence and severity):
See below
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
See below
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): See below
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
See below
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
In life examinations
No effect of treatment with TCP was evident from mortality data. One female (animal number 339) in the 20 mg/kg/day group was found dead on Gestation Day (GD) 4. Clinical findings seen in this animal included rapid breathing (GO 2-3), red material around nose (GD 1-3), skin cold to touch (GD 1-3), skin discolored red on the forepaws (GD 1-2). At necropsy, adhesions involving the heart, lungs and thoracic cavity, and a lung mass were noted. These were considered suggestive of a dosing injury. All remaining animals in the control and treated groups survived to scheduled euthanasia.
No effect of treatment at the 20 mg/kg/day dose level was evident from clinical evaluations. Findings seen in these animals occurred at low incidence or with similar frequency as controls and were considered unrelated to treatment. Salivation was seen with increased frequency in groups treated with TCP at dose levels of 100,400, and 750 mg/kg/day. Salivation was seen at least once during the daily clinical examinations in 11, 24, and 25 animals in the 100, 400, and 750 mg/kg/day groups, respectively. It was not seen among the control or 20 mg/kg/day animals. Other clinical findings seen with increased frequency in the 400 and 750 mg/kg/day groups and considered related to treatment were sparse amounts of hair in the abdominal and lumbar regions, ventral surface and hind limbs, and unkempt appearance.
No effect of treatment with TCP at dose levels of 20 and 100 mg/kg/day was evident from gestation body weights or body weight gain. At the 400 and 750 mg/kg/day dose levels, body weights on GD 20 were statistically lower than controls and body weight gain was statistically lower than controls over GD 15-18, 18-20, and 0-20. A dose-responsiveness was evident in these data and the 750 mg/kg/day group actually experienced a mean weight loss of 0.3 grams over GD 18-20 in comparison to a 35 gram weight gain in controls. The 400 mg/kg/day animals experienced a weight gain of 22 grams over this period. Weight gain over the entire GD 0-20 period was 126.5 grams in the 400 mg/kg/day group, about 14% lower than controls (146.6 grams), and 94.9 grams in the 750 mg/kg/day group, a 35% reduction from controls.
No effect of treatment with TCP at dose levels of20 and 100 mg/kg/day was evident from gestation food consumption data. No clear effect on gestation food consumption was evident at the 400 mg/kg/day dose level, but at 750 mg/kg/day, food consumption was statistically lower than controls over GD 0-3 and 18-20. The lower food consumption over this latter interval (15.7 g/animal/day vs.24.1 g/animal/day in controls) was consistent with the weight loss for this group over this interval. Noteworthy from the food consumption data for these treatment groups (400 and 750 mg/kglday) was the increased spillage seen among these animals. Eleven animals (44%) in the 400 mg/kg/day group and 10 animals (40%) in the 750 mg/kg/day group were noted with excessive spillage of feed at one or more recording intervals over the 20-day gestation period. Similar spillage was not seen among the 25 control females or among the females in the 20 and 100 mg/kg/day groups. The increased frequency of spillage at these dose levels appeared to be treatment-related but its toxicological significance, if any, is unclear.
Postmortem study evaluations
No effect of treatment was evident from maternal macroscopic examinations. The few macroscopic findings seen among the treated animals occurred at low incidence and were considered unrelated to treatment.
Pregnancy rates ranged from 92 to 100% among all the groups and provided 24, 23, 24, 25, and 25 GD 20 litters for evaluation in the control, 20, 100, 400, and 750 mg/kg/day groups, respectively. No effect of treatment with TCP at a dose level up to and inclusive of750 mg/kg/day was evident from uterine implantation data. The mean number of corpora lutea, uterine implantation sites, fetuses, and resorption sites in the treated groups was comparable to controls. Likewise, the mean pre- and post-implantation loss indices for the treated groups were comparable to controls.
Gravid uterine weights, adjusted GD 20 body weights, and adjusted body weight gain over GD 0-20 were comparable to controls in the 20 and 100 mg/kg/day groups. In the 400 and 750 mg/kg/day groups, gravid uterine weights were statistically lower than controls. Adjusted GD 20 body weights and adjusted body weight gain (GD 0-20) were also lower than control in these same groups; however, only at the 750 mg/kg/day dose level were these differences statistically significant. The lower gravid uterine weights in the 400 and 750 mg/kg/day groups were consistent with lower fetal body weights.

Effect levels (maternal animals)

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

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Fetal body weights in the treated groups were lower than controls. At the 20 and 100 mg/kg/day dose levels these differences were slight (-4.4 to -6.3%) in comparison to controls and of greater magnitude, -9% and -18% in the 400 and 750 mg/kg/day groups, respectively. In most cases these differences in fetal weights, distinguished by sex and for both sexes combined, were statistically significant in comparison to controls. A dose-response analysis (linear trend test) of fetal body weights was also conducted for males, females, and pooled (combined) sexes. For all of these comparisons (males, females, pooled sexes), the trend analyses revealed a significant linear pattern in the data across treatments (p=<0.0001). Mean fetal body weights in the treated groups were outside the low range of recent historical control data for this laboratory, while mean fetal body weights for controls were within this historical range. The lower fetal body weights seen in the treated groups were considered indicative of a treatment-related response.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): See below
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See below
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See below
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See below
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
See below
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No effect of treatment with TCP was evident from the fetal skeletal malformation data. The few skeletal malformations seen among fetuses in the 100, 400, and 750 mg/kg/day groups occurred at low incidence and were considered spontaneous and unrelated to treatment. Skeletal malformations of the head (squamosal misshapen and small mandible) were seen in the one fetus in the 100 mg/kg/day group noted externally with mouth, jaw, and eye malformations. In the 400 mg/kg/day group, one fetus was noted with skeletal malformations involving the head (jugal absent and squamosals small). In the 750 mg/kg/day group, absent rib and defects of the lumbar and thoracic vertebrae were seen in one fetus. No skeletal malformations were seen among fetuses in the control and 20 mg/kg/day group. A delay in ossification, as indicated from an increase in unossified/incompletely ossified bones (i.e., ossification variations), was apparent at the 750 mg/kg/day dose level. The incidence of litters containing at least one fetus with an ossification variation in this group was 100%, which differed statistically from the 75% incidence in controls. There was also an increase in litter incidence of incompletely ossified skull bones (occipitals, frontals, parietals, and supraoccipitals) and unossified sternebrae in this group, consistent with a delay in ossification, but only for the latter was the difference from controls statistically significant. At the lower dose levels, some variability was seen in the litter incidence of several ossification variations and in the incidence of litters containing fetuses with variations, but these differences from controls were not statistically significant, and no clear effect of treatment was evident.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
See below
Visceral malformations:
no effects observed
Description (incidence and severity):
See below
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal body weights in the treated groups were lower than controls. At the 20 and 100 mg/kg/day dose levels these differences were slight (-4.4 to -6.3%) in comparison to controls and of greater magnitude, -9% and -18% in the 400 and 750 mg/kg/day groups, respectively. In most cases these differences in fetal weights, distinguished by sex and for both sexes combined, were statistically significant in comparison to controls. A dose-response analysis (linear trend test) of fetal body weights was also conducted for males, females, and pooled (combined) sexes. For all of these comparisons (males, females, pooled sexes), the trend analyses revealed a significant linear pattern in the data across treatments (p=<0.0001). Mean fetal body weights in the treated groups were outside the low range of recent historical control data for this laboratory, while mean fetal body weights for controls were within this historical range. The lower fetal body weights seen in the treated groups were considered indicative of a treatment-related response.
No effect of treatment with TCP at a dose level up to and inclusive of 750 mg/kg/day was evident from fetal sex ratios (% male fetuses/litter). Mean sex ratios in the treated groups ranged from 44.2 to 53.7% and were considered comparable to the 47.6% in controls.
No effect of treatment with TCP was evident from fetal external examinations. Gastroschisis was seen in one fetus in the 20 mg/kg/day group (litter incidence 4.3%) and with edema in three fetuses from a single litter in the 400 mg/kg/day group (litter incidence 4.0%). Gastroschisis has not been noted in recent historical control data for the laboratory, but in the absence of this or similar malformations among the 750 mg/kg/day fetuses, its occurrence in this study was considered spontaneous and unrelated to treatment. Malformations of the mouth, jaw, and eyes were seen in one fetus in the 100 mg/kg/day group. In the absence of similar malformations among fetuses at the higher dose levels, this too was considered a spontaneous occurrence and unrelated to treatment. No malformations were secn in the control and 750 mg/kg/day fetuses at exteral examination.
No effect of treatment with TCP was evident from the fetal visceral examinations. No malformations were seen at visceral examination of the control and treated fetuses. Increased renal pelvic cavitation, a visccral variation, was seen with low incidence of occurrence in the control, 100, 400, and 750 mg/kg/day groups. These incidences in the treated groups were comparable to controls and no effect of treatment was evident. This finding was not seen among fetuses in the 20 mg/kg/day group.
No effect of treatment with TCP was evident from the fetal skeletal malformation data. The few skeletal malformations seen among fetuses in the 100, 400, and 750 mg/kg/day groups occurred at low incidence and were considered spontaneous and unrelated to treatment. Skeletal malformations of the head (squamosal misshapen and small mandible) were seen in the one fetus in the 100 mg/kg/day group noted externally with mouth, jaw, and eye malformations. In the 400 mg/kg/day group, one fetus was noted with skeletal malformations involving the head (jugal absent and squamosals small). In the 750 mg/kg/day group, absent rib and defects of the lumbar and thoracic vertebrae were seen in one fetus. No skeletal malformations were seen among fetuses in the control and 20 mg/kg/day group. A delay in ossification, as indicated from an increase in unossified/incomplete1y ossified bones (i.e., ossification variations), was apparent at the 750 mg/kg/day dose level. The incidence of litters containing at least one fetus with an ossification variation in this group was 100%, which differed statistically from the 75% incidence in controls. There was also an increase in litter incidence of incompletely ossified skull bones (occipitals, frontals, parietals, and supraoccipitals) and unossified sternebrae in this group, consistent with a delay in ossification, but only for the latter was the difference from controls statistically significant. At the lower dose levels, some variability was seen in the litter incidence of several ossification variations and in the incidence of litters containing fetuses with variations, but these differences from controls were not statistically significant, and no clear effect oftreatment was evident.
No adverse effect of treatment with TCP at a dose level up to and inclusive of 750 mg/kg/day was evident from fetal malformation data. The overall incidence of litters containing at least one fetus with a malformation from either the external, visceral, or skeletal examinations was low in each of the treated groups (ranging from 4.0 to 4.3%) and did not differ statistically from the 0% incidence in controls.

Effect levels (fetuses)

Key result
Dose descriptor:
LOAEL
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: see explanation below

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
Treatment related:
no
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
yes

Any other information on results incl. tables

The mean fetal body weights and litter sizes were collected from each dam. As outlined in the protocol, the analysis for fetal bodyweights included tests to determine if it was appropriate to conduct an analysis of covariance using litter size as a covariate. If the assumptions for the covariate failed, the model was run with just treatment as an effect and a follow-up group pair-wise analysis was run. If the assumptions did not fail, the model was run with treatment and litter size and pairwise comparisons were made using Dunnett's test. Upon examination of the data from the above analysis, a follow-up trend analysis was requested. A dose-response analysis (linear trend test) was conducted using linear contrasts under the appropriate statistical model. The dose-response analysis was run for males, females and pooled sexes.

 

Males: The assumptions on the analysis of covariance were violated and thus, pair-wise comparisons were performed without the covariate. Treatments 2, 4 and 5 were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattem in the data across treatments (p = <0.0001).

 

Females: The assumptions on analysis of covariance were met and thus, an analysis of covariance was performed with the litter size as the covariate. Pair-wise comparisons showed that all treatments were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattern in the data across treatments (p = <0.0001).

 

Males and Females Combined: The assumptions on analysis of covariance were violated and thus, pair-wise comparisons were performed without the covariate. All treatments were significantly different from the control with respect to the mean fetal body weights. Furthermore, a trend analysis revealed a significant linear pattern in the data across treatments (p<0.0001).

Applicant's summary and conclusion

Conclusions:
In this oral rat developmental toxicity study with tricresyl phosphate (TCP), the No-Observable-Effect Level (NOEL) for maternal toxicity was 20 mg/kg/day. At 100 mg/kg/day, the only maternal toxicity seen was an increased frequency of salivation following dosing. At the higher dose levels evaluated (400 and 750 mg/kg/day), the frequency of animals with sparse amounts of hair in the abdominal and lumbar regions, ventral surface, and hind limbs, and unkempt appearance was also increased. Lower body weights and body weight gain during gestation were seen at 400 and 750 mg/kg/day and reduced food consumption was seen at 750 mg/kg/day. Excessive feed spilage was also seen with increased frequency among females in the 400 and 750 mg/kg/day groups. No effect of treatment was evident from maternal macroscopic findings, uterine implantation data, fetal sex ratios, or fetal malformation data. Lower fetal body weights were seen at all dose levels evaluated. An increase in variations during the skeletal examinations indicative of a delay in fetal ossifìcation was seen at 750 mg/kg/day. Thus, the 20 mg/kg/day dose level was determined to be the Lowest-Observable-Adverse-Effect Level (LOAEL) for developmental toxicity as a NOEL could not be determined due to decreased fetal body weights in all treated groups.
Executive summary:

In this oral rat developmental toxicity study with tricresyl phosphate (TCP), the No-Observable-Effect Level (NOEL) for maternal toxicity was 20 mg/kg/day. The 20 mg/kg/day dose level was also determined to be the Lowest-Observable-Adverse-Effect Level (LOAEL) for developmental toxicity as a NOEL could not be determined due to decreased fetal body weights in all treated groups.