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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Rationale: GLP Guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(see Principles of Method)
Principles of method if other than guideline:
This protocol exceeded the OECD 422 study design by extending the F1 offspring to adulthood (Day 70), with continued exposure and assessments of neurologic, immunologic and reproductive structures and functions. The protocol also assessed recovery in F0 males and females exposed for 28 days.
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): Triphenyl Phosphite (TPPi); identified by the supplier, Dover Chemical
Corporation (Dover, OH), as Doverphos 10
- Physical state: clear to light-colored liquid
- Analytical purity: 99.7%
- Lot/batch No.: 237T03101
- Stability under test conditions: TPPi was shown to be stable in com oil for at least 35 days under refrigerated conditions and stable for 4 hours under simulated dosing conditions.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, NC
- Age at first dose: (F0) 10 wks; (F1) 3 wks
- Housing: The animals were 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 with Sani-Chip® cage litter. Study animals were housed 2 per cage (1 male:l female from the same dose group) during the mating period. 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 Fl weanlings (1 O/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) was available ad libitum.
- Water (e.g. ad libitum): Tap water was available ad libitum
- Acclimation period: approx. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 66-77°F (22±3°C)
- Relative Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12-hour light cycle per day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: dosing formulations were prepared in corn oil at concentrations of 1, 3, and 8 mg/ml approximately every 30 days and stored under refrigeration.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 refomrnlated 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.
Duration of treatment / exposure:
F0 males: 28 days
F0 females: 10 weeks
F0 recovery females: 28 days
F1 offspring: 7 weeks postweaning
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 5, 15 and 40 mg/kg/day
Basis:

No. of animals per sex per dose:
F0 males: 10 per group
F0 recovery males: 5 controls and 5 high dose
F0 females: 10 per group
F0 28 day females: 5 controls and 5 high dose
F0 recovery females: 5 contols and 5 high dose
F1 offspring: 10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Original doses were chosen based on a rangefinding (RF) study. The definitive study was restarted due to unanticipated excessive toxicity seen at doses of 200, 100 and 50 mg/kg/day.

Examinations

Observations and examinations performed and frequency:
Parental animals: Observations and examinations

Observations for mortality were made twice daily (a.m. and p.m.), and the general condition of all animals was checked daily. Clinical examinations were conducted and recorded daily throughout the course of the study.

A FOB, including home cage observations, handling observations, open field observations, sensory and neuromuscular observations, and physiological observations, 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.

The body weights of the F0 male rats were determined and recorded initially and then weekly throughout mating. Body weights of the 28-day females and recovery males and females were recorded on a weekly basis until termination. The body weights of F0 female rats were recorded in the same manner until confirmation of mating. During gestation, F0 females were weighed on gestational days (gd) 0, 7, 14, and 20. Dams producing litters were weighed on pnd 0, 4, 7, 14, and 21. Body weight gains were computed.

Feed consumption measurements were recorded weekly for all F0 parental animals during the 2-week prebreed exposure period and recorded weekly for the 28-day females. Feed consumption collection periods corresponded with the collection of the animals' weekly body weights. During pregnancy of F0 females, feed consumption was recorded for gd 0-7, 7-14, and 14-20. During lactation of Fl litters, maternal feed consumption was measured for pnd 0-4, 4-7, 7-14, and 14-21, although maternal feed consumption after pnd 14 was confounded by the contribution from the pups, since pups are self-feeding by this time. Feed consumption was not measured during the period of cohabitation, since 2 adult animals (1 male and 1 female) were in the same cage. Feed consumption was not measured for the recovery animals during or post dosing.

Five F0 males per group, randomly selected at the end of the mating period, and the 28-day females, prior to necropsy, were singly housed overnight in metabolism cages. The total amount of time the animal was in the chamber and the amount of urine collected were recorded. The urine was evaluated for appearance and by· dipstick analysis for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocytes.

Hematology and blood chemistry was evaluated on 5 randomly selected FO males per group and from the 5 females in the high-dose and control groups on the last day of the pre breed period (study day 13). Hematology parameters measured included 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. 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) was conducted on clinical chemistry blood samples.
Sacrifice and pathology:
Postmortem examinations (Parental animals)

All F0 parental animals, 28-day females from the control and high-dose groups, as well as recovery males and females were subjected to a complete grossnecropsy, with selected organs weighed and retained. Uteri of F0 females were examined for the number of nidation (implantation) scars.

Full histopathology was performed on all retained organs from 5 randomly selected F0 males and females (and the 28-day F0 females) in the control and high-dose groups, with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. If treatment-related changes were observed in the high-dose group, then histopathologic examinations were extended to the mid- and low-dose group animals. The affected organs from the recovery animals were also examined histopathologically, if appropriate.

Postmortem examinations (Offspring)

All nonselected Fl weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. The retained F1 offspring were sacrificed at approximately70 days of age and subjected to the same assessments as the F0 parents (except for evaluation of nidation scars, which are not relevant to the Fl females because they are not mated)

Full histopathology was performed on all retained organs from 5 randomly selected Fl males and females in the control and high-dose groups, with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. If treatment-related changes were observed in the high-dose group, then histopathologic examinations were extended to the mid- and low-dose group animals. Because of toxicity in the F1 offspring at 40 mg/kg/day, no F1 offspring were retained after weaning. Therefore, tissues from 5 F1 males and females per group at 0 and 15 mg/kg/day were examined histopathologically.

Other examinations:
The OECD 422 study design (OECD, 1996) specifies termination of the study on pnd 4, with external and internal examination of the Fl pups at this time. This modified study design provided for continuation of the Fl offspring, with continuing exposure until sexual maturity.

At weaning, at least 1 female and 1 male (whenever possible) from each Fl litter, for a total of 10/sex/group, were selected on a random basis to continue treatment for approximately 7 more weeks (dosing for Fl 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 Fl 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.

Fl postweaning observations and procedures for each retained female included examination for VP (from pnd 22 until acquisition of vaginal opening) and determination of estrous cyclicity and normality, evaluated by vaginal smears taken daily the last 3 weeks of the postwean exposure period prior to scheduled sacrifice. For each retained male offspring, observations for PPS began at 35 days of age and continued until acquisition of PPS.

Body weights were recorded on the day of acquisition of VP and PPS. All retained F 1 weanlings were weighed and feed consumption measured once per week until their scheduled demise. Daily Fl mortality and clinical observations were conducted as described for the F0 animals.

FOBs were performed on 5 randomly selected postwean Fl offspring/sex/group once, midway through the postwean period, as described for the F0 parental animals. Grip strength was determined in the last week of the postwean period for the same 5 animals/sex/group used for FOB. The procedures were the same as those described for the F0 generation.

In addition, blood was collected, and hematology, clinical chemistry (Fl males and females), and urinalysis (Fl males only) conducted on 5 randomly selected Fl animals/sex/group prior to necropsy, as described for the F0 males and 28-day females.

All nonselected Fl weanlings were subjected to a complete gross necropsy (external and visceral) examination, with gross lesions retained in 10% neutral buffered formalin. The retained F 1 offspring were sacrificed at ~ 70 days of age and subjected to the same assessments as the F0 parents (except for evaluation of nidation scars, which are not relevant to the Fl females because they are not mated).
Statistics:
The unit of comparison was the male, female, pregnant female, or the litter, as appropriate. Treatment groups were compared to the concurrent control group using either parametric ANOV A under the standard assumptions or robust regression methods (Zeger and Liang, 1986; Royall, 1986; Huber, 1967).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
TPPi, administration resulted in adult F0 parental toxicity at 40 mg/kg/day, increasing over time (reduced body weight gain, ataxia, and foot splay). There were no other findings attributed to the test substance. Reproductive toxicity was not present in F0 males or females.

There was profound Fl offspring toxicity observed postnatally during lactation at 40 mg/kg/day. So this group was terminated on pnd 21 due to increased mortality, especially for pnd 0-4, and reduced pup body weights/litter starting on pnd 7 through 21. F1 offspring in other groups were weaned at Day 22 and then treated with TPPi for 7 weeks (until postpartum day 70). For these retained Fl males and females, in-life systemic parameters were unaffected, with acquisition of puberty in both sexes and andrological parameters in adult F1 males also unaffected in the surviving dose groups.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEL
Remarks:
P systemic toxicity
Effect level:
15 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: lower body weight gain, ataxia, and foot splay at 40 mg/kg/day
Dose descriptor:
NOAEL
Remarks:
F1 pups, lactation phase
Effect level:
15 other: mg/kg/day (maternal dose)
Sex:
male/female
Basis for effect level:
other: increased mortality, reduced pup body weight/litter at 40 mg/kg/day (maternal dose)
Dose descriptor:
NOAEL
Remarks:
F1 adults
Effect level:
15 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

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 effects during lactation were 15 mg/kg/day for males and females; these effects are considered to be secondary to severe maternal toxicity or of the same nature as the adult toxicity observed at this dose, and is not considered to be indicative of a specific effect by TPP on development. The F1 male and female systemic NOAEL was also 15 mg/kg/day.
Executive summary:

TPPi was administered by gavage once daily at 0, 5, 15, and 40 mg/kg/day to parental F0 CD® (SD) rats, 1 O/sex/group, through prebreed, mating, gestation, and lactation and direct dosing to Fl offspring (10/sex/group) from weaning to postpartum day 70. Treatment with TPPi resulted in adult F0 parental toxicity at 40 mg/kg/day, increasing over time (reduced body weights, ataxia, and foot splay). Twenty-eight-day males (same as F0) and females and 28-day recovery males and females exhibited the same progressive systemic toxicity at 40 mg/kg/day as in the F0 parental animals, although the effects were less severe, most likely due to the shorter dosing duration. Reproductive toxicity was not present in F0 males or females.

 

There was profound Fl offspring toxicity observed postnatally during lactation at 40 mg/kg/day. So this group was terminated on pnd 21 due to increased mortality, especially for pnd 0-4, and reduced pup body weights/litter starting on pnd 7 through 21. F1 offspring in other groups were weaned at Day 22 and then treated with TPPi for 7 weeks (until postpartum day 70). For these retained Fl males and females, in-life systemic parameters were unaffected, with acquisition of puberty in both sexes and andrological parameters in adult F1 males also unaffected in the surviving dose groups.

 

Based on these results, the F0 male and female systemic no observable adverse effect level (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 NOAEL for Fl offspring effects during lactation were 15 mg/kg/day for males and females. The Fl adult male and female systemic NOAEL was also 15 mg/kg/day.