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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:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Rationale: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 422
Deviations:
yes
Remarks:
This protocol exceeded the OECD 422 study design by following the F1 offspring to adulthood, with continued exposure and assessments of neurologic, immunologic and reproductive structures and functions. The protocol also assessed F0 recovery males.
Principles of method if other than guideline:
This protocol exceeded the OECD 422 study design by following the F1 offspring to adulthood, with continued exposure and assessments of neurologic, immunologic, and reproductive structures and functions. The protocol also assessed F0 recovery males, 28-day females, and 28-day recovery females.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TPPi
- Physical state: clear to light-colored liquid
- Analytical purity: 99.7%
- Lot/batch No.: Doverphos 10, Batch No. 237T03101
- Supplier: Dover Chemical Corporation (Dover, OR).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC (males and females both from Area R12)
- Age at study initiation: ~63 days old
- Weight at study initiation: Males 321.2 - 362.9 g; Females 216.3 - 252.6 g.
- Fasting period before study:
- Housing: All animals were housed in the RTI Animal Research Facility (Room 303) for the duration of the study. Individually housed upon arrival, during the acclimation period, and upon the initiation of the treatment period in solid-bottom polycarbonate cages with stainless-steel wire lids (Laboratory Products, Rochelle Park, NJ) with Sani-Chip® cage litter (P.J. Murphy Forest Products Corp., Montville, NJ). Females were caged individually once they were successfully mated (or at the end of the mating period) and throughout gestation. Females were housed with their litters throughout the lactation period. Randomly selected F1 weanlings (10/sex/group), males, and females were singly housed during the postweaning exposure period.
- Diet (e.g. ad libitum): Pelleted Purina Certified Rodent Diet® (No. 5002, PMI Feeds, Inc., St. Louis, MO; batch numbers documented in the study records) ad libitum.
- Water: Tap water (source: City ofDurham, Department of Water Resources, Durham, NC) ad libitum in plastic water bottles with butyl rubber stoppers and stainless-steel sipper tubes.
- Acclimation period: ~ 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70%
- Photoperiod: 12-hour light cycle per day
- There were no temperature or RH deviations outside the ranges specified in the protocol during the conduct of the study

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Mazola Oil
Details on exposure:
oral gavage with an appropriate-sized syringe fitted with a 16-g, 3-inch (for adults) or 18-g 112 -inch (for weanlings) stainless-steel, curved dosing needle (Perfektum®, Popper and Sons, New Hyde Park, NY).

PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:


VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the start of the study, homogeneity of TPPi in corn oil, storage stability, and stability under conditions simulating the dosing procedures were conducted at concentrations of 1 and 10 mg/ml TPPi in corn oil. TPPi was shown to be stable in corn oil for at least 35 days under refrigerated conditions and stable for 4 hours under simulated dosing conditions. Therefore, dosing formulations were prepared in corn oil at concentrations of 1, 3, and 8 mg/ml approximately every 30 days and stored under refrigeration. All dosing formulations were analyzed for concentration verification.

Standards for acceptable accuracy of mixing were the mean of the analyzed samples was within ± 10% of nominal concentration, and the % RSD (Relative Standard Deviation) for triplicate samples did not exceed 10%. All study formulations met these standards except for the 1, 3, and 8 mg/ml doses formulated on July 10, 2003. When samples taken from the dosing bottles were analyzed, the 1.0 mg/ml formulation was acceptable. The 3.0 mg/ml sample was reformulated on July 14,2003, and was acceptable for use. The 8.0 mg/ml sample was not reformulated since this dose group was discontinued on June 18,2003
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
2 weeks ofprebreed exposure (males and females), 2 weeks of mating (males and females), and 3 weeks of gestation and lactation each (F0 females) for F0 parents, and direct dosing of selected F1 offspring from weaning through scheduled sacrifice, at least 7 weeks postweaning.
Frequency of treatment:
once daily, 7 days per week
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes
Details on study design:
- Groups: 4 groups, 10 F0 males/group and 10 F0 females/group, to yield at least 8 pregnant females/group at or near parturition, 5 males and 5 females/group for the control and high-dose groups designated as recovery animals, and 5 females each for the control and high-dose groups to be terminated at the end of the 28-day dosing period, for a total of 110 animals; 50 males and 60 females.
- F0 animals were uniquely identified prior to initiation of the study by ear tag. Selected F1 weanlings were uniquely identified by eartag.
- Dose selection rationale: rangefinding (RF) study, employing doses of 0, 100, 300, and 1000 mg/kg/day, (oral gavage) once a day to 5 animals/sex/group at 5 ml/kg for 10 consecutive days.
- Rationale for animal assignment: Animals were assigned by sex to the different groups by means of randomization stratified by body weight, such that the body weights by sex of all groups were homogeneous at treatment initiation.
- Other: Five additional F0 males per group from the control and 40 mg/kg/day groups were designated as recovery animals and held without dosing for 2 weeks after the F0 male dosing period was completed, to evaluate recovery from any possible treatment-related effects identified in the high-dose group. Additional 28-day females (5/group at 0 and 40 mg/kg/day) and 28-day recovery females (5/group at 0 and 40 mg/kg/day) were also similarly assessed.

F1 Group
- At weaning, at least 1 female and 1 male (whenever possible) from each F1 litter, for a total of 10/sex/group, were selected on a random basis to continue treatment for 7 more weeks (dosing for F1 selected pups began on pnd 22 and continued until all pups were at least 70 days of age). All pups were available for selection except those not expected to survive because of physical abnormalities. Records were maintained on any pup excluded from the selection process. Any F1 pup that appeared moribund or that died during lactation was necropsied, when possible, to investigate the cause of death and to identify internal visceral developmental malformations, if any. Organs were not weighed for these animals.
-

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: mortality, twice daily (a.m. and p.m.), general condition, daily.
- Cage side observations checked included: changes in skin and fur, eyes, mucous membranes, respiratory and circulatory systems, autonomic and central nervous systems, somatomotor activity, and behavior pattern. Beginning on gd 20, each female was observed twice daily (a.m. and p.m.) for evidence of littering.

DETAILED CLINICAL OBSERVATIONS: Yes, Prior to necropsy (sd 28), hematology was performed on 5 randomly selected F0 males per group and from the 5 females in the high-dose and control groups on the last day of the prebreed period (sd 13).
- Paremeters checked: evaluation of hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, RBC indices, and prothrombin time (PT), a measure of blood clotting time/potential (Becton Dickinson Fibrometer). Evaluation in serum of sodium, potassium, chloride, glucose, total cholesterol, blood urea nitrogen (BUN), creatinine, total protein and albumin, and 2 enzymes indicative of hepatocellular effects (alanine aminotransferase and aspartate aminotransferase) were done on the clinical chemistry samples.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males recorded initially and weekly through mating; 28-day females and recovery males and females
were recorded on a weekly basis until termination; of F0 female rats were recorded in the same manner until confirmation of mating; During gestation on days (gd) 0,7, 14, and 20. Dams producing litters were weighed on pnd 0,4,7,14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, except during the period of cohabitation. Feed consumption was not measured for the recovery animals during or post dosing.
- Time schedule for examinations: weekly for all F0 parental animals during the 2-week prebreed exposure period and recorded weekly for the 28-day females. During pregnancy of F0 females, feed consumption was recorded for gd 0-7, 7-14, and 14-20. During lactation ofFllitters, maternal feed consumption was measured for pnd 0-4, 4-7, 7-14, and 14-21

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes, all adult animals were euthanized by C02 asphyxiation.
- Sacrifice on gestation day #
- Organs examined:

OTHER: A FOB, including home cage observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations (Moser, 1989; Moser et aI., 1991), was performed on F0 males and 28-day females prior to dosing and weekly to termination at sd 28. A FOB was also performed on F0 females prior to dosing and weekly during prebreed, mating, gestation, and lactation and on recovery animals (male and female) prior to dosing, weekly during dosing, and once during the recovery period.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes / No / No data
- Number of corpora lutea: Yes / No / No data
- Number of implantations: Yes
- Number of early resorptions: Yes / No / No data
- Number of late resorptions: Yes / No / No data
- Other:
Fetal examinations:
On the day of birth (pnd 0), anogenital distance was measured and body weight recorded for all live Fl pups in all litters. Body weight was recorded for all live pups on pnd 4 prior to culling and euthanasia. Anogenital distance was recorded with the individual pup weight on pnd 0. The presence or absence ofretained nipples and areolae on the ventrum was recorded for F1 offspring males at pnd 11-13. F1 offspring males, with 1 or more retained nipples on pnd 11-13, were uniquely marked on the tail within litters. All pups were examined for physical abnormalities (external developmental malformations) at birth and throughout the preweaning and postwean periods.
- Sacrifice: F1 pups culled on pnd 4 were sacrificed by decapitation.
- F1 postweaning observations and procedures for each retained female included examination for VP (from pnd 22 until acquisition of vaginal opening) and determination of
Statistics:
Unit of comparison was the male, female, pregnant female, or the litter, as appropriate. Treatment groups were compared to the concurrent control group using parametric ANOVA under standard assumptions or robust regression methods, the latter of which do not assume homogeneity of variance or normality. The homogeneity of variance assumption was examined via Levene's Test. If (p<0.05), robust regression methods were used to test all treatment effects. They were used in this study to test for overall treatment group differences (via Wald Chi-Square Test), followed by individual t-tests for exposed vs. control group comparisons when the overall treatment effect was significant. If Levene's Test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied to comparing the treatment groups. The GLM procedure in SAS® Release 8 was used to evaluate the overall effect of treatment and, when a significant treatment effect was present, to compare each exposed group to the control via Dunnett's Test. For the litter-derived percentage data (e.g., periodic pup survival indices), the ANOVA was weighted according to litter size. A one-tailed test was used for all pairwise comparisons to the vehicle control group, with the exception that a two-tailed test was used for parental and pup body weight and organ weight parameters, feed consumption, percent males per litter, and anogenital distance. Student's t-test was used to analyze body weight and organ weights from the recovery males and females and the 28-day females. Reproductive indices were analyzed using Chi-Square Test of Independence. When p<0.05 Fisher's Exact Test, with adjustments for multiple comparisons, was used to compare each treatment group to controls. Acquisition of developmental landmarks and anogenital distance, were analyzed using ANCOVA as well ANOVA.
Indices:
Survival indices were calculated on pnd 0,4, 7, and 14 and at weaning. There was no mortality and no effects on male or female reproductive indices. Precoital interval and gestational length were also unaffected. There were no effects on prenatal (postimplantation) loss or on total or dead pups per litter in any group on pnd 0 or 21.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
All of the 28-day females and the 28-day recovery females (each with 5/group at 0 and 40 mg/kg/day) survived to scheduled necropsy.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
>= 40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: other:
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
toxicity during lactation
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
Systemic
Effect level:
15 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Numbers of live pups per litter were significantly reduced on pnd 0 at 40 mg/kg/day. All pups in 2 litters died at 40 mg/kg/day.

Effect levels (fetuses)

Dose descriptor:
LOAEL
Effect level:
40 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

No parental males died on study. All 5 recovery males each at 0 and 40 mg/kg/day survived to scheduled sacrifice. F0 male body weights were equivalent for all time points for sd 0, 7, 14, 21, and 28 in all groups. During the prebreed and mating periods (sd 0 to 28) , F0 male body weight change was significantly decreased at 40 mg/kg/day for sd 14-21 and 21-28 and at 15 mg/kg/day for sd 21-28, and unaffected for all other intervals at these doses and for all intervals at 5 mg/kg/day. Feed consumption values, expressed as g/day and g/kg/day, were significantly increased at 15 mg/kg/day for sd 7 to 14, with no effects at 5 or 40 mg/kg/day. Clinical observations of F0 males during this period included alopecia (limbs) in 2, 1, 2, and 2 males each at 0, 5, 15, and 40 mg/kg/day, respectively, and rooting postdosing in 1 male at omg/kg/day and in 4 males at 40 mg/kg/day (likely due to taste aversion, commonly observed in gavage dosing, and not toxicity, per se). Efflux of dosing solution was observed in 1, 4, 2, and 2 males at 0, 5, 15, and 40 mg/kg/day, respectively. The only findings considered treatment- and dose-related ataxia observed in 5 males at 40 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
The F0 male and female systemic no observable adverse effect (NOAEL) was 15 mg/kg/day. The NOAELs for F0 reproductive toxicity were at or above 40 mg/kg/day for males and females. The NOAELs for F1 offspring toxicity during lactation were 15 mg/kg/day for males and females. The F1 male and female systemic NOAEL was also 15 mg/kg/day.