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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: pellets
Details on test material:
- Name of test material (as cited in study report): Triphenyl phosphate
- Physical state: White pellets
- Analytical purity: 99.5%
- Lot/batch No.: CH13/085
- Stability under test conditions: stable
- Storage condition of test material: Room temperature

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Nulliparous, non-pregnant and untreated females were used at inititation of the study.
- Age at delivery: 17-19 weeks; mating with adult and proven fertile males at WIL Research
- Fasting period before study: none
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% aqueous solution
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was grinded to a fine powder in a grinding mill. Subsequently, the powder was suspended in the vehicle. Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 1% aqueous carboxymethyl cellulose was chosen based on trial formulations performed at WIL.
- Amount of vehicle (if gavage): 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (26 November 2014), according to a validated method (Project 506608). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (lowest concentration only).
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
a) A copulation plug was observed, and / or
b) The daily vaginal smear was sperm positive.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used only for mating. These male rats are in the possession of RCC and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.
Duration of treatment / exposure:
Day 6 - 28 post coitum inclusive
Frequency of treatment:
once daily
Duration of test:
All animals surviving to the end of the observation period (Day 29 post-coitum) and the female with premature delivery were euthanised by intravenous injection of pentobarbital (approx. 1 mL/kg Euthasol®20%) and subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs and the fetuses.
No. of animals per sex per dose:
Each group consisted of 22 mated female rabbits.
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose levels
Dose levels were selected based on the results of a dose range finding study (Project 505943; see APPENDIX 6). In this latter study dose levels of 83, 250 and 750 mg/kgmg/kg bw/day were tested. Due to severe toxicity, all females at 750 mg/kgmg/kg bw/day and one female at 250 mg/kgmg/kg bw/day had to be euthanized before scheduled necropsy. Another female at 250 mg/kgmg/kg bw/day was noted with clinical signs (pale appearance on Days 22 and 23 post-coitum and reduced production of (pale) faeces from Day 17 post-coitum). She had no food consumption from Days 16-23 post-coitum and body weight loss (up to -6%). No signs of toxicity were noted for the remaining females at 250 mg/kgmg/kg bw/day and all females at 83 mg/kgmg/kg bw/day. Based on these data, dose levels of 32, 80 and 200 mg/kgmg/kg bw/day were selected for the present prenatal developmental toxicity study.

Examinations

Maternal examinations:
Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals.
All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups.
Ovaries and uterine content:
The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated.
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or represent slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized by administration of approximately 0.3 mL (= 60 mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) into the oral cavity using a small flexible plastic or metal feeding tube. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed.

Visceral (Internal):
All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by Stuckhardt and Poppe (Ref. 1). This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was determined by internal examination.

The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. The heads from the remaining one-half of the fetuses in each litter of all groups were examined by a mid-coronal slice.

All carcasses, including the carcasses without heads, were eviscerated, skinned and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses.

The specimens of all groups will be archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.

A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
For each litter the following calculations were performed:

Pre-implantation loss (%) = (number of corpora lutea - number of implantation sites) divided by the number of corpora lutea x 100
Post-implantation loss (%) = (number of implantation sites - number of live fetuses) divided by the number of implantation sites x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:

Viable fetuses affected/litter (%) = number of viable fetuses affected/litter divided by the number of viable fetuses/litter x 10
Historical control data:
available

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
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): one femaled in the high dose group delivered early
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination) in the tested doses of up to and including 200 mg/kg bw/day. Higher doses were shown to induce strong toxicity (mortality at 750 and 250 mg/kg bw/day) in the dose range finding studies and were thus not included in the main study.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
>= 200 mg/kg bw/day (actual dose received)
Basis for effect level:
other: developmental toxicity

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment at 200 mg/kg bw/day resulted in a higher incidence of lungs with absent accessory lung lobe(s). While in the control and mid dose groups only one fetus (A015-03 and A048-02, respectively) each was noted with this malformation, the incidence increased to 3(3) fetuses (litter) in the high dose
group (A067-03, A069-07, A074-04). This resulted in a litter proportion of 1.6% which was at the upper limit of the available historical control range for this finding (MAX: 1.7%). In addition, 2 dead fetuses from litter 78 that were delivered preterm on Day 29 post-coitum (A078-03 and A078-05) had absent
accessory lung lobes. The total litter proportion of fetuses at 200 mg/kg bw/day with absent accessory lung lobe(s) thus increased from 1.6% (given in Table 1.15; APPENDIX 1) to 2.4%. This litter proportion was clearly above the range of available historical control data. Lungs with absent accessory lung lobe(s) is a more common finding in New Zealand White rabbits. The historical control data from this laboratory consisted of 17 developmental studies with this strain in which in total 2787 (315) control fetuses (litters) were examined. In 10 of these studies fetuses with absent accessory lung lobe(s) were found, i.e. in total 20 (17) control fetuses (litter). The highest incidence was 3(3) fetuses (litter) seen in 2 studies. As in the high dose group of the present study the incidence of lungs with absent accessory lobe(s) was only slightly higher with in total 5(4) fetuses (litter) including the 2 fetuses from the preterm delivered litter, it was considered as an incidence finding and thus not to be toxicologically relevant.
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
There were no developmental findings up to and including 200 mg/kg bw/day that were considered to be toxicologically relevant.
No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations). Higher doses were shown to induce strong toxicity (mortality at 750 and 250 mg/kg bw/day) in the dose range finding studies and were thus not included in the main study.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Applicant's summary and conclusion

Executive summary:

Triphenyl phosphate was tested in a prenatal developmental toxicity study in pregnant New Zealand rabbits following OECD TG 414. The test item was administered once daily by oral gavage from Days 6 to 28 post-coitum at doses of 0, 32, 80 and 200 mg/kg bw/day in 1% aqueous carboxymethyl cellulose. The doses were chosen on the basis of dose range finding studies that showed strong toxicity (including mortality) at 750 and 250 mg/kg bw/day.

Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Formulations prepared on one day during treatment were analyzed for accuracy, homogeneity and stability.

All animals surviving to Day 29 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative. All fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol. The fetuses of all groups were stained with Alizarin Red S for skeletal examinations.

No toxicologically significant changes were noted in any of the maternal parameters investigated in this study (i.e. mortality, clinical signs, body weights, food consumption, and macroscopic examination). No toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external, visceral and skeletal developmental malformations or variations, visceral variations).

Based on the results of this prenatal developmental toxicity study, both the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for triphenyl phosphate were established as being at least 200 mg/kg bw/day, since no adverse effect was observed. No higher doses could be tested in pregnant rabbits based on dose range finding studies.