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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
liquid
Details on test material:
Batch no: CH2/23017

Tris(1-chloroprop-2-yl) phosphate, isomer mixture (CAS 13674-84-5): 78.0 %
Bis(1-chloropro-2-yl) 2-chloropropyl phosphate, isomer mixture (CAS 76025-08-6): 19.6 %
1-Chloroprop-2-yl bis(2-chloropropyl) phosphate, isomer mixture (CAS 76649-15-5): 1.8 %
Tris(2-chloropropyl) phosphate, isomer mixture (CAS 6145-73-9): 0.06 %
Sum of (Chloropoxy)propyl bis(chloropropyl) phosphates [= 'TCPP ether']: 0.3 %
Sum of unknown components (5), major unknown component: 0.1 %
Water content by Karl Fischer: < 0.1 %
Residue of evaporation: < 0.1 %
Sum of all: 100.0 %
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lab specific test material number: 208366/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in refrigerator
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stability was analytically confirmed for at least 5 hours at room temperature under normal laboratory light conditions, 13 days in the refrigerator, and 3 weeks in the freezer (≤ -15°C) over the concentration range 1 to 200 mg/mL. Test Facility Study No. 517747 (ABL study no.17117).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: none

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Chatillon sur Chalaronne, France
- Untreated females from a non-inbred laboratory colony were mated at the supplier and were at day 0 or 1 post-coitum on arrival at the Test Facility (day 0 post-coitum is the day of successful mating)
- Age at delivery: 18-20 weeks; a health inspection was performed upon receipt of the animals
- Fasting period before study: none
- Housing: individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): relative target humidity of 40 to 70%; the actual daily mean temperature during the study period was 18 to 20°C with an actual daily mean relative humidity of 58 to 90%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES Main Study:
From: 29 September 2017 To: 11 October 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose with 0.1% Tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 5 hours after adding the vehicle to the test item or were prepared weekly by formulating daily portions which were stored in the refrigerator. When prepared weekly, dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water was chosen based on trial formulations performed at Charles River and information provided by the Sponsor.
- Amount of vehicle (if gavage): 5 ml/kg bw
- Preparation of vehicle: The vehicle, 1% Aqueous carboxymethyl cellulose with 0.1% Tween 80, was prepared at least monthly, stored in a refrigerator set to maintain 4°C. The prepared vehicle was removed from the refrigerator and stirred for at least 30 minutes before dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by using a validated analytical procedure (ABL No. 17118).
Concentration analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. The concentrations analysed were in agreement with the target concentrations. No test item was detected in the control group formulation.
Homogeneity analysis: Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was below or equal 10%. The formulations of group 2 and 4 were homogeneous.
Stability analysis: Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 517747, ABL No. 17117) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 517747.
Details on mating procedure:
Untreated females were mated at the Supplier, and were at Day 0 or 1 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).
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) 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.
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
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 (Test Facility Study No. 517745) and in an attempt to produce graded responses to the test item.
The dose levels for this study were selected based on the results of a tolerability study in non-pregnant rabbits by oral gavage in which no mortality or severe clinical signs became obvious up to and including 1000 mg/kg bw/day (Test Facility Study No. 517744).
In the dose range finding study dose levels of 500, 750 and 1000 mg/kg bw/day were tested in 6 pregnant rabbits per dose and control from gestational day 6 to 28 by oral gavage.
Treatment at 1000 mg/kg bw/day group was poorly tolerated by pregnant females and resulted in early termination of this group. Four out of six females (nos. 19-22) were euthanized in extremis on Days 12 or 13 post-coitum. From treatment onwards (Day 6 postcoitum), these females lost about 280-560 gram body weight (i.e. 9-14%) and had significantly reduced or no food consumption. Moreover, severely reduced feces production piloerection. At necropsy, one female was emaciated and one female had cysts on her ovary and scab formations. Due to animal welfare reasons, the remaining two females (nos. 23-24) of this dose group were sacrificed on Day 13 post-coitum as well.
One female at 750 mg/kg bw/day (no. 16) was euthanized on Day 25 post-coitum as she delivered her offspring early. This female showed a body weight loss of 9% from treatment onwards and had no food consumption over Days 15-24 post-coitum. A lean appearance, reduced feces production, dark urine and alopecia were noted in addition. No abnormalities were observed at necropsy. No toxicologically relevant findings were noted for the remaining animals treated up to 750 mg/kg bw/day.
Based on the results of this dose range finder, in which four animals of the 1000 mg/kg bw/day group and one animal of the 750 mg/kg bw/day group showed severe general toxicity (e.g. severely reduced food consumption, body weight loss) selected dose levels for the main study were 75, 200 and 500 mg/kg bw/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 18, 21, 24, 27, and 29 post-coitum

FOOD CONSUMPTION: Yes
- Days 2-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29; females with early delivery (nos. 76 and 83): within 24 hours of early delivery
- Examination at necropsy: All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution).
- Organ weight: liver weight
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes (not for premature decedents)
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: pre- and post- implanation loss
Fetal examinations:
Live fetuses were euthanized by administration of sodium pentobarbital (Euthasol® 20%) into the oral cavity using a small metal feeding tube. Fetuses of premature decedents were externally examined in detail and euthanized if necessary by decapitation or by administration of sodium pentobarbital.

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 into the oral cavity using a small flexible plastic or metal feeding tube. Recognizable fetuses of females that were killed in extremis were examined externally. Nonviable fetuses (the degree of autolysis was minimal or absent) were examined and weighed. For late resorptions a gross external examination was performed (if possible). Late resorptions with malformations were fixed in 10% buffered formalin.

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. 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. 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 for subsequent examination of skeletons.

Skeletal:
The eviscerated fetuses from all groups, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S by a method similar to that described by Dawson. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination was not extended to the fetuses from the low and mid dose group.

The specimens of all groups were archived in glycerin with bronopol 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:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as
indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but
excluded semi-quantitative data, and any group with less than 2 observations.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric: Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
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 were compared using the Mann Whitney test.
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.
Incidence:
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.

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:
Maternal variables:
Body Weight Gains: Calculated between at least each scheduled interval.
Corrected Body Weight Gains: Terminal body weight minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the Terminal body weight.

Reproduction and development variables - 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 100
Historical control data:
historical control data of fetal examination are given for values that are outside the expected range or of statistical significance, as appropriate

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Description (incidence and severity):
Clinical signs:
No clinical signs were noted for surviving females that were considered to be toxicologically relevant.
Reduced feces production up to a severe degree was observed for almost all animals in all groups, including controls. This finding is commonly observed for rabbits of this age and strain which are housed and treated under the conditions in this study, and as no dose-related trend was observed, this was not considered to be toxicologically relevant.
A lean appearance, diarrhea, red discoloration of the genital region and red fluid on manure tray were noted for single females across the groups during the treatment period. Restless behavior was noted once for two females treated at 200 mg/kg bw/day, of which one female showed a cramped posture as well, and for one female at 500 mg/kg bw/day. One female at 500 mg/kg bw/day was noted on one single day during the treatment period with rales and a bleeding nose. As these findings occurred incidentally and did not persist over time, they were not considered to be toxicologically relevant.
For clinical signs noted for premature decedents in the low and mid dose group (75 and 200 mg/kg bw/day) see section 'mortality' and Table 1
Description (incidence):
Mortality:
see Tables 1 and 3: Four females of each the low and mid dose group (75 and 200 mg/kg bw/day) died or were euthanized in extremis within a few seconds to minutes after the oral gavage procedure between Day 6 and Day 23 post-coitum.
One female (no. 38) was not dosed on the day of euthanasia as difficulties were noted when inserting the gavage tube. For three females (nos. 38, 53 and 60) blood was noted at the end of the gavage tube. For the other females, no difficulties or abnormalities were noted during the gavage procedure. Clinical signs that were observed directly after the gavage procedure included labored respiration (three females), gasping (two females), clonic spasms (four females), a pale appearance and uncoordinated movements (one single female). In addition, three females were noted with a bleeding nose and/or mouth. These effects were almost directly followed by death or euthanasia. Macroscopic findings were mainly observed in the respiratory tract of these premature decedents. Foamy contents in the trachea were noted for four females. Reddish foci in the lungs (up to many) were noted for seven females, of which two females especially showed foci in the right caudal lung lobe. Three females showed inflation of the lungs. For single females each, a ruptured right caudal lobe, reddish discolored lungs or a perforation of the right caudal lung lobe was noted. Additionally, hemorrhagic and clotted blood in the thoracic cavity or a hardened heart were noted for one female each.
In conclusion, these 8 unscheduled deaths occurred only in the low and mid dose group, and not in the high dose group, and the moribund effects occurred timely very closely related to the gavage procedure, i.e. within a few seconds to minutes. Most of the clinical signs (blood at the end of the gavage tube, gasping, labored respiration) and macroscopic findings in the lungs and trachea can be related to the oral gavage procedure, and for two out of eight females a perforation or ruptured lung lobe were observed at necropsy indicating a misapplication. Therefore, the deaths can be interpreted as a consequence of the gavage procedure and are not likely attributed to toxicological effects of the test item.
One female at 500 mg/kg bw/day (no. 83) was euthanized on Day 22 post-coitum, as she delivered her offspring early.
Description (incidence and severity):
see Table 2 - body weight and body weight changes:
Mean body weights were slightly lower in the 200 and 500 mg/kg bw/day groups compared to controls during the treatment period, but changes were not statistically significant. At the end of the treatment period, mean body weights at 200 and 500 mg/kg bw/day were 1% and 4%, respectively, lower than controls.
Mean body weight gains were up to two times lower at 200 and 500 mg/kg bw/day compared to controls, reaching statistical significance on Days 9, 15, 21 and 24 post-coitum in the 200 mg/kg bw/day group and on Days 9 to 21 post-coitum in the 500 mg/kg bw/day group.
In addition, mean body weight gains corrected for gravid uterus were slightly lower at 200 and 500 mg/kg bw/day than controls (-5.3% compared to -2.3% in the control group), but remained within the range considered normal for rabbits of this age and strain.
The marginal changes at 75 mg/kg bw/day compared to controls remained within the normal range and were not considered to be toxicologically relevant.
Description (incidence and severity):
Food consumption - treatment related effect:
Treatment at 500 mg/kg bw/day resulted in statistically significantly reduced food consumption before and after correction for body weight from Day 6 to 21 post-coitum. After correction for body weight, differences of minus 20 % (days 6-9 and 9-12 pc), minus 26% (days 12-15 pc), minus 23% (days 15-18 pc), and minus 18% (days 18-21 pc) were noted compared to controls. Thereafter, no significant changes were recorded. Remark: see also chapter 'maternal toxicity: other effects'
At 200 mg/kg bw/day, statistically significantly lower values for food consumption were noted over Days 12-15 post-coitum (absolute only) and Days 18-21 post-coitum (both absolute and relative). Over Days 18-21 post-coitum, a relative difference of 18% was noted compared to controls.
During the remaining treatment period, food consumption values of treatment groups remained in the same range as controls.
No toxicologically relevant changes were noted at 75 mg/kg bw/day.
Description (incidence and severity):
Organ weight findings:
In the 75 and 500 mg/kg bw/day groups, mean liver weight and liver/body weight ratio were higher than controls. Relative differences in ratio were 9% and 14% respectively over control, reaching statistical significance at 500 mg/kg bw/day.
The individual liver weight of female no. 70, noted with multiple liver abnormalities, remained within the normal range.
Description (incidence and severity):
Gross pathological findings:
There was an increased incidence of (dark) reddish foci on the lungs in the treatment groups compared to the control group. In total, including the macroscopic findings of the 8 premature decedents (see Table 1), lung foci were observed for 1, 8, 4 and 4 females in the control, 75, 200 and 500 mg/kg bw/day group, respectively.
One female at 500 mg/kg bw/day (animal no. 70) was noted with multiple liver abnormalities, including dark reddish discoloration and an irregular surface of the right lateral and caudate lobes and the caudate lobe grew together with the lobus dexter lateralis. As these findings were observed for a single female, they were considered to be caused by chance and of no toxicological relevance.
Description (incidence and severity):
justification for dose selection: The high dose of 500 mg/kg bw/day was considered to be the highest tolerated dose, as in the dose range finder treatment at 750 mg/kg bw/day resulted in severe toxic effects for 1/6 females, including a 9% body weight loss and absence of food consumption. Moreover, treatment at 1000 mg/kg resulted in unacceptable toxicity (i.e. body weight loss of 9-14%, absence of food consumption, piloerection and/or a lean/pale appearance) for 4/6 females followed by early euthanasia on Days 12-13 post-coitum.

Maternal developmental toxicity

Description (incidence and severity):
see Table 3 - number of abortions:
One female at 500 mg/kg bw/day (no. 83) delivered her offspring on Day 22 post-coitum. This female had a body weight loss of 5% over Days 6-21 post-coitum and a significant reduced or no food consumption over Days 15-21 post-coitum. In the animal facility, five out of ten fetuses were noted to be alive, while at necropsy all fetuses were dead. All ten fetuses were noted to be relatively small (weight was not determined). For five of them, severe external malformations were observed, including gastroschisis, omphalocele and absence of skin on the head. Remark: see also chapter 'maternal toxicity - other effects'
Description (incidence and severity):
pre and post-implantaion loss
no adverse effects observed - see Table 4: No toxicologically relevant effects on the numbers of corpora lutea and implantation sites,and pre- and post-implantation loss were noted by treatment up to 500 mg/kg bw/day.
Description (incidence and severity):
Total litter losses by resorption:
no adverse effects observed - see Tables 3 and 4
Description (incidence and severity):
Early or late resorptions:
no adverse effects observed - see Table 4
Description (incidence and severity):
see Table 4 - Dead fetuses:
Two fetuses in the 500 mg/kg bw/day group (A075-08 and A076-02) were dead at scheduled necropsy.
Description (incidence and severity):
changes in pregnancy duration:
no effects observed
Migrated Data from removed field(s)
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Effects on pregnancy duration:
no adverse effects observed; For one female at 500 mg/kg bw/day (no. 76) early delivery was noted in the morning of scheduled necropsy on Day 29 post-coitum. She delivered six pups of which one pup was dead and partly cannibalized. As no abnormalities were noted for this female, this is not considered to be toxicologically relevant.
Description (incidence and severity):
see Table 3 - changes in the number of pregnants:
There were one control female and two females each at 75, 200 and 500 mg/kg bw/day which were not pregnant. Pregnancy could not be determined for one female at 200 mg/kg bw/day (no. 53) which was moribund on Day 6 post-coitum. No implantation sites or corpora lutea were detectable for this female at necropsy, which was due to the early stage of pregnancy. This female is reported as non-pregnant in the data tables.
Description (incidence and severity):
Maternal developmental toxicity - other effects:
In one litter which was early delivered on Day 22 post-coitum, two fetuses with gastroschisis, two fetuses with omphalocele of the trunk and one fetus without skin on its head were noted and all fetuses were noted to be relatively small of size. It can be excluded that these findings were caused by cannibalism as no torn or bleeding skin layer was noted. The respective dam showed signs of toxicity; body weight loss of 5% over Days 6-21 post-coitum and a significant reduced or no food consumption over Days 15-21 post-coitum. This could indicate a delayed developmental stage of the fetuses. It is known that during embryo-fetal development of rabbits, a physiological umbilical hernia occurs which starts around gestation Day 12.5 to 14.5 and is reduced around gestation Day 20 (DeSesso, J.M. Coparative features of vertebrate embryology. Chapter 6 of Developmental reproductive toxicology: a practical approach by Taylor & Francis Group LLC, 2016). Moreover, when evaluating available historical data sets, it showed that omphalocele and/or umbilical hernia occur at an overall litter incidence of 0.3-1.1% with a lack of dose-responsiveness, and that maternal toxicity or stress (decreased food consumption and/or weight gain) may play a role in the cause of omphalocele (Daston, G., Beekhuijzen, M. Is omphalocele a non-specific malformation in New Zealand White rabbits? Reproductive Toxicology, 78, 29-39, 2018). It might be possible that at this developmental stage, mechanical forces during the early delivery process (i.e. adhesion of amniotic membranes) could have contributed to the observed wall defects. It is thus uncertain whether these malformations were related to the maternal toxicity in combination with the developmental stage of the fetuses and/or mechanical forces during the early delivery process. The early delivered fetuses were not taken into account when determining the developmental NOAEL.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: the NOAEL is the highest dose tested

Maternal abnormalities

Abnormalities:
no effects observed
Localisation:
other: Transient but significantly reduced body weight gain from days 9-21 pc and reduced food consumption from days 6-21 pc were recorded. Liver weight increased at 500 mg/kg bw by 14%. These observations were not considered as adverse.

Results (fetuses)

Description (incidence and severity):
see Table 5 - fetal body weight changes:
Fetal body weights were lower at 500 mg/kg bw/day compared to controls. Mean combined fetal weights were 41.8, 41.3, 39.9, and 38.1 g for the control, 75, 200 and 500 mg/kg bw/day groups, respectively. At 500 mg/kg bw this weight was about 9% lower than controls. The mean values were lower than the 5th percentile of the available historical control data. However, these changes were not statistically significant and as ossification parameters were unaffected, indicating no growth retardation effects, this was considered to be non-adverse.
Male fetal weights were also lower at 75 ( and 200 mg/kg bw/day compared to the concurrent control group. The changes in these groups were considered to be caused by the relatively high control value, which was higher than the 95th percentile of the available historical control data and mainly caused by one control litter (no. A009; four male fetuses with a mean body weight of 57.6 gram). Therefore, this was not considered to be toxicologically relevant.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see Tables 4 and 5: The numbers of alive fetuses (litters) available for fetal morphological examination were 183 (21), 134 (16), 150 (16) and 177 (19) in Groups 1, 2, 3, and 4, respectively.
Two fetuses in the 500 mg/kg bw/day group (A075-08 and A076-02) were dead at scheduled necropsy. As fetus A076-02 was partly cannibalized, no morphological examination was performed.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see Tables 4 and 5: The male:female ratio was unaffected by treatment up to 500 mg/kg bw/day. Mean sex ratios (males:females) were 45:55, 47:53, 50:50 and 49:51 for the control, 75, 200 and 500 mg/kg bw/day groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see Table 4: There were no treatment-related effects on litter size of any group. Mean litter sizes were 8.7, 8.4, 9.4 and 9.3 viable fetuses/litter for the control, 75, 200 and 500 mg/kg bw/day groups, respectively.
Description (incidence and severity):
see Table 6 - external malformations:
At scheduled necropsy, in one litter at 500 mg/kg bw/day one fetus (dam A068 - fetus no. 15) was found with multiple malformations, including exencephaly, gastroschisis, carpal flexure and ectrodactyly, which were, where possible, also skeletally confirmed to detect an underlying skeletal origin. During skeletal examination, sternoschisis was noted for this fetus additionally. Such a combination of malformations is not uncommon for incidentally appearing multiple malformed rabbit fetuses. Although gastroschisis was not seen previously in historical controls of the Test Facility, these malformations were regarded as an incidental finding and considered not to be related to treatment with the test item as they were observed in one single fetus at scheduled necropsy only. The other 14 fetuses in the litter did not show any abnormalities.
Other externally malformed fetuses were two control fetuses of the same litter (A015-1 and - 8) that had carpal flexures without an underlying skeletal origin and one group 2 (75 mg/kg bw) fetus (A036-8) with omphalocele. These malformations were noted previously in historical controls at comparable frequencies. As the carpal flexures observed in one litter were noted in the control group, they were not test item related. The omphalocele observed in Group 2 was regarded to be caused by chance as one single litter was affected without any other malformations noted in the low and mid dose group.
External variations were not observed in this study (see Table 7).
Description (incidence and severity):
see Table 6 - skeletal malformations:
There were no treatment related effects on skeletal morphology following treatment up to 500 mg/kg bw/day.
Skeletal malformations in fetuses of which the dam received test item were sternoschisis (Group 4 fetus A068-15 with multiple external malformations) and vertebral anomaly with associated rib anomaly (Group 4 fetuses A076-06 and A080-03).
Vertebral anomaly with or without associated rib anomaly was also observed in three fetuses of the control group (A002-04, A007-10 and A014-02) and was noted previously in historical controls. Therefore, this malformation was regarded to be unrelated to treatment.

see Table 7 - skeletal variations:
There was a statistically significantly increased litter incidence of fetuses with 13th rudimentary in Group 4 (500 mg/kg bw), compared to the concurrent control group. This variation was observed at mean litter incidences of 3.8% and 10.6% per litter in Groups 1 and 4, respectively (Groups 2 and 3 were not examined skeletally). This statistical significance was considered to have arisen as a result of a remarkably low control value, which was lower than the minimum historical control incidence of 4.8%. Moreover, the Group 4 value remained within the range of historical control data and therefore, these changes were not considered to be treatment-related.
Other skeletal variations that were noted in this study occurred at low incidences, in the absence of a dose-related incidence trend and/or at frequencies that were within the range of available historical control data.
Description (incidence and severity):
see Table 6 - visceral malformations:
There were no treatment-related effects on visceral morphology following treatment up to 500 mg/kg bw/day.
Two visceral malformations occurred in this study. Tetralogy of Fallot was observed in two fetuses from the same litter of Group 3 (A048-5 and -9) and in one control fetus (A022-2).
Internal hydrocephaly was observed in one fetus of Group 4 (A070-1) during the Wilson sectioning technique. Tetralogy of Fallot is a commonly observed finding among historical controls. Also the internal hydrocephaly was observed previously in historical control fetuses with a maximum historical litter incidence of 0.8% versus 0.5% noted in this study. In absence of a dose-related trend and as the occurrence was within the range of available historical control data, these were considered to be chance findings and not toxicologically relevant.

see Table 7 - visceral variatioins:
The total incidence of visceral variations per litter was statistically significantly increased at 500 mg/kg bw (13.4% versus 4.6% in controls, 6.8% at 75 mg/kg bw and 6.8% at 200 mg/kg bw). This was caused by an increased incidence of two variations, i.e. retrocaval ureter and absent accessory lung lobe (both not statistically significant). Mean litter incidences of retrocaval ureter were 2.1, 2.5, 2.3 and 6.0 % in Groups 1, 2, 3 and 4, respectively. In comparison to the maximum available historical control incidence of 6.3%, the increase at 500 mg/kg bw was considered not to be toxicologically relevant. In addition, the incidence of absent accessory lung lobe was increased without statistical significance in Groups 3 and 4, compared to controls. Mean litter incidences were 0.0, 1.0, 2.3 and 2.3% in Groups 1, 2, 3 and 4, respectively. As this variation was observed previously in historical control fetuses (maximum historical control value of 1.7 %) and is one of the most commonly observed variations in rabbits, these changes in Groups 3 and 4 were considered to be unrelated to treatment.
All other variations noted, were not considered treatment-related as they occurred infrequently, and/or occurred at frequencies that were within the range of the available historical control data.
Description (incidence and severity):
In this study, severe malformed fetuses were noted in one early delivered litter on Gestation Day 22 at 500 mg/kg. However, it is uncertain whether these malformations were related to the maternal toxicity in combination with the developmental stage of the fetuses and/or mechanical forces during the early delivery process. Therefore, the early delivered fetuses were not taken into account when determining the developmental NOAEL. (see also: maternal developmental toxicity - other effects)

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: the NOAEL is the highest dose tested

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Overview of premature decedents directly (within a few seconds to minutes) after the oral gavage treatment procedure

 Group  Animal No.  Day of necropsy  Termination  Clinical signs  Macroscopic findings
 75 mg/kg bw/day  25  day 23 post-coitum  spontaneous death  bleeding nose and labored respiration

 trachea: foamy contents

lungs: many reddish foci

   37  day 21 post-coitum  sponatneous death  clonic spasms and labored respiration

 lungs: ruptured right caudal lobe and several reddish foci

thoracic cavity: hemorrhagic / clotted blood

   38  day 07 post-coitum  euthanized in extremis  blood at the end of tube, bleeding nose and gasping  lungs: several dark reddish foci, reddish discoloration and inflation
 43  day 20 post-coitum  spontaneous death  bleeding nose and clonic spasms

 trachea: foamy contents

lungs: several dark reddish foci in the right caudal lobe

 200 mg/kg bw/day  47  day 13 post-coitum  spontaneous death  clonic spasms

 heart: hardened

trachea: foamy contents

lungs: isolated reddish foci and inflation

   53  day 06 post-coitum  euthanized in extremis

 blood at the end of tube, bleeding nose/mouth,

labored respiration, uncoordinated movements and

pale appearance

lungs: inflation  
   60  day 19 post-coitum  spontaneous death  blood at the end of tube and gasping

 lungs: perforation of the right caudal lobe, many dark reddish

foci and foamy contents

   64  day 20 post-coitum  spontaneous death  clonic spasms

 trachea: foamy contents

lungs: many dark reddish foci on the right caudal lobe

Table 2: Body weights (in g, rows 1 and 2) and body weight gain (in %, rows 3 -10) of dams

  means  control group  75 mg/kg bw/day  200 mg/kg bw/day  500 mg/kg bw/day
  day 2 pc (body weight in g)  3425  3441  3410  3386
  day 29 pc  3986  3948  3937  3819
  day 9 pc (body weight gain in %)  2  1  1*  0**
  day 12 pc  4  3  2  2*
  day 15 pc  8  5  4**  4**
  day 18 pc  8  7  5  5*
  day 21 pc  9  8  5*  5*
  day 24 pc  10  8  6*  8
  day 27 pc  11  9  8  8
  day 29 pc  12  10  9  9

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3: Summary of maternal survival and pregnancy status

  Dose group  control     75 mg/kg    200 mg/kg    500 mg/kg  
   No.  %  No.  %  No.  %  No.  %
 Females on study  22    22    22    22  
 Females that aborted or delivered  0 0.0   0  0.0  0  0.0  1  4.5
 Females that died  0 0.0  4 18.2  4  18.2  0  0.0
 Females that aborted  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  1  25.0  0  0.0
 Gravid  0  0.0  4  100.0  3  75.5  0  0.0
 Females that were euthanized  0  0.0  0  0.0  0  0.0  0  0.0
 Nongravid  0  0.0  0  0.0  0  0.0  0  0.0
 Gravid  0  0.0  0  0.0  0  0.0  0  0.0
 Females examined at scheduled necropsy#  22  100 .0  18  81.8  18  81.8  21  95.5
 Nongravid  1  4.5  2  11.1  2  11.1  2  9.5
 Gravid  21  95.5  16  88.9  16  88.9  19  90.5

 with resorptions only

 0  0.0  0  0.0  0  0.0  0  0.0
 with viable fetuses  21  100.0  16  100.0  16  100.0  19  100.0
 Total females gravid  21  95.5  20  90.9  19  86.4  20  90.9

#- Including females that delivered Day 29: A076 (500 mg/kg bw/day)

Table 4: Summary of fetal data at scheduled necropsy

 Group  

Sex

 M

Sex 

F

 Viable

fetuses

 Dead

fetuses

Resorptions

 Early

 Resorptions

Late

 Post

implantation

loss

 Implantation

sites

 Corpora

lutea

 Pre

implantation

loss

Fetal

weights

in grams 

 No. of

gravid

females

 control  Total 83  100  183  0  4  10  14  197  212  15  NA  21
   Mean  4.0  4.8  8.7  0.0  0.2  0.5  0.7  9.4  10 .1  0.7  41.8  
 75 mg/kg  Total  65  69  134  0  6  3  9  143  149  6  NA  16
   Mean  4.1  4.3

 8.4

 0.0

 0.4

 0.2

 0.6

 8.9

 9.3

 0.4

 41.3

 

 200 mg/kg

 Total

 73

 77

 150

 0

 5

 4

 9

 159

 160

 1

 NA

 16

 

Mean 

 4.6

 4.8

 9.4

 0.0

 0.3

 0.3

 0.6

 9.9

 10.0

 0.1

 39.9

 

 500 mg/kg

 Total

 86

 91

 177

 2

 9

 2

 13

 190

 197

 7

 NA

 19

 

 Mean

 4.5

 4.8

 9.3

 0.1

 0.5

 0.1

 0.7

 10.0

 10.4

 0.4

 38.1

 

None significantly different from control group

NA = not applicable

Mean number of viable fetuses, mean number of implantation sites, mean number of corpora lutea,

Fetal weights compared using Dunnett's test

Table 5: Summary of fetal data at scheduled necropsy [% per litter]

Group 

 0 mg/kg

 75 mg/kg

 200 mg/kg

 500 mg/kg

 Number of litters

 21

 16

16

 19

Corpora lutea

 

 

 

 

 Mean

 10.1

 9.3

 10.0

 10.4

 Implantation sites

 

 

 

 

 Mean

9.4

 8.9

 9.9

 10.0

 Viable fetuses (%)

 

 

 Mean

 93.4

 93.7

 94.5

 93.4

 Dead fetuses (%)

 

 

 

 Mean

 0.0

 0.0

 0.0

 1.3

 Early resorptions (%)

 

 

 

 

 Mean

 2.8

 4.0

 3.2

 4.5

 Late resorptions (%)

 

 

 

 Mean

 3.8

 2.3

 2.2

 0.8

 Total resorptions (%)

 

 

 

 

 Mean

 6.6

 6.3

 5.5

 5.3

 Pre-implantation loss (%)

 

 

 

 

 Mean

 7.2

 3.6

 0.7

 3.6

 Post-implantation loss (%)

 

 

 

 

 Mean

 6.6

 6.3

 5.5

 6.6

 Males (%)

 

 

 

 

 Mean

 44.7

 47.4

 49.8

 49.0

 Females (%)

 

 

 

 

 Mean

 55.3

 52.6

 50.2

 51.0

 Male fetal weights (g)

 

 

 

 

 Mean

 42.6

 40.4

 39.7

 38.4

 Female fetal weights (g)

 

 

 

 

 Mean

 40.6

 41.2

 40.0

 37.6

 Combined fetal weights (g)

 

 

 

 

 Mean

 41.8

 41.3

 39.9

 38.1

Proportional (%) data compared using the Mann-Whitney test

Fetal weights compared using Dunnett's test

None significantly different from control group

Table 6: Summary of fetuses and litters with variations [absolute no.]

 

 Fetuses    

 Litters    

 Dose group (mg/kg bw per day):

 0

 75

200

500

 0

 75

 200

 500

 Number examined externally

 183

 134

 150

 177

 21

 16

 16

 19

exencephaly

 0

 0

 0

 1#

 0

0

 0

1

gastroschisis

 0

 0

 0

 1#

0

0

1

1

carpal and/or tarsal flexure

 2

 0

 0

 1#

1

 0

0

1

 omphalocele

 0

 1

 0

 0

0

1

0

0

 ectrodactyly

 0

 0

 0

 1#

0

0

0

1

Number examined viscerally

 183

 134

 150

 177

21

16

16

19

 teratology of Fallot

 1

 0

 2

 0

 1

 0

 1

 0

hydrocephaly - internal

0

 0

 0

 1

 0

0

0

1

 Number examined skeletally

 183

 0

 0

 177

 21

 0

0

 19

 vertebral anomaly with or without associated rib anomaly

 3

 -

 -

 2

 3

 -

 -

 2

 sternoschisis

 0

 -

 -

 1#

 0

 -

 -

 0

 Total number with malformations

 

 

 

 

 

 

 

 

 external

 2

 1

 0

 1

 1

 1

 0

 1

 soft tissue

 1

 0

 2

 1

 1

 0

 1

 1

 skeletal

 3

 0

 0

 3

 3

 0

0

 3

 combined  6  1  2  4  5  1  4

# fetus A068 -15 with multiple malformations

Table 7: Summary of fetuses and litters with variations

 Dose group  control  75 mg/kg

 200 mg/kg

 500 mg/kg

 

 No.

 litters

 No.

litters

 No.

 litters

 No.

 litters

 Number examined externally

 183

 21

 134

16

 150

16

177

19

 number with findings externally

 0

0

 0

0

 0

0

 0

 0

Number examined viscerally

183

21 

 134

16 

 150

16

177

19

 retrocaval ureter

 4

3

 3

3

3

1

10

8

 retrocaval ureter (mean litter incidence)

 

2.1%

 

2.5%

 

2.3%

 

6.0%

lung- absent accessory lobe

 0

0

1

1

3

2

4

3

lung- absent accessory lobe (mean litter incidence)

 

0.0%

 

1.0%

 

2.3%

2.3%

 Number examined skeletally

 183

21

-

-

 -

-

 177

19

 13th rudimentary rib(s)

 8

6

 -

 -

 -

 -

18

12

 13th rudimentary rib(s) (mean litter incidence)

 

3.8%

 -

 -

 -

 -

 

10.6%*

 Total variations

 

 

 

 

 

 

 % per litter with external variations

 

0.0

 

 0.0

 

 0.0

 

 0.0

 % per litter with soft tissue variations

4.6

 

 6.8

 

 6.8

 

13.4*

 % per litter with skeletal variations

71.6

 

 -

 

 -

 

73.4

 total % per litter with variations

 

 72.9

 

 

 

 

 

77.0

* = Significantly different from the control group at 0.05

Applicant's summary and conclusion

Executive summary:

The objectives of this study were to determine the potential of the registered substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to 22 time-mated female New Zealand White rabbits per dose group from Day 6 to 28 post-coitum, inclusive. The study was performed according to OECD TG 414.

The dose levels in this study were selected to be 0, 75, 200, 500 mg/kg bw/day, based on the results of the dose range finder in which all animals of the 1000 mg/kg bw/day group and one animal of the 750 mg/kg bw/day group showed severe signs of general toxicity (e.g. severely reduced to absence of food consumption, body weight loss). The high dose of 500 mg/kg bw/day was thus considered to be the highest tolerated dose.

According to the results in the main OECD 414 prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Reaction products of phosphoryl trichloride and 2-methyloxirane (TCPP) was established as being at least 500 mg/kg bw/day, the highest dose level tested.