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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 20, 2014 to September 02, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): PETIA
- Lot/batch No.: JBJL0043T
- Physical state: Clear colourless viscous liquid
- Expiration date of the lot/batch: 25 March 2016
- Storage Conditions: At room temperature, protected from light

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: 17-19 weeks
- Housing: individually housed in labelled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days prior to pairing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at WIL Research Europe
- Amount of vehicle (if gavage): 1 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 (10 July 2014). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature under protection from light was also determined (highest and lowest concentration).

Duplicate samples (approximately 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 20 mL. For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations. For determination of stability, additional samples were taken at 50% height and stored at room temperature protected from light for 6 h. The volumetric flasks were filled up to the mark with acetonitrile. The solutions were further diluted to obtain an end solution of 25/75 (v/v) acetonitrile/water and concentrations within the calibration range. All solutions containing the test substance were protected from light.

Analytical conditions:
- Instrument: Acquity UPLC system (Waters, Milford, MA, USA)
- Detector: Acquity UPLC TUV detector (Waters)
- Column: Acquity UPLC BEH Shield RP-18, 100 mm × 2.1 mm i.d., dp = 1.7 μm (Waters)
- Column temperature: 40°C±1°C
- Injection volume: 10 μL
- Mobile phase: 30/70 (v/v) acetonitrile/water
- Flow 0.45 mL/min
- UV detection: 225 nm


The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Details on mating procedure:
One female was placed on a one-to-one-basis in the cage of a male rabbit. The time of mating was established by visual observation of mating. This day was designated Day 0 post-coitum.
Duration of treatment / exposure:
From Day 6 to Day 28 post-coitum, inclusive.
As a misgavage was suspected, female no. 45 (Group 3) was not dosed on Days 13 and 14 post-coitum. This animal died on Day 16 post-coitum.
Frequency of treatment:
Once daily for 7 days/week, approximately the same time each day
dose.
Duration of test:
From Day 6 to Day 28 post-coitum, inclusive
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 50 and 75 mg/kg bw/day
Basis:

No. of animals per sex per dose:
24, 22, 26 and 24 females in groups 1, 2, 3 and 4, respectively; group 1 being control
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were selected based on the results of a dose range finding study. In this study, six mated rabbits per groups were dosed at 0, 25, 50 or 75 mg/kg bw/day for Days 6 to 28 postcoitum,inclusive, by oral gavage. At 75 mg/kg bw/day, toxicity consisted of reduced faeces production, reduced body weight gain (with a body weight loss on Days 6 to 9 post-coitum) and reduced food consumption on Days 6 to 9 post-coitum. At 75 mg/kg bw/day, one female had dark red fluid in the uterus. This animal had 5 live fetuses and 6 early resorptions, which resulted in a slightly increased post-implantation loss at this dose. At all dose levels, fetal body weights were slightly lower compared to controls, but this was not statistically significant and did not show a dose relationship.

Examinations

Maternal examinations:
MORTALITY/VIABILITY:
At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ (2000)7). The circumstance of any death was recorded in detail.

CLINICAL SIGNS:
At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) were scored. Cage debris was examined in case of abortions.

BODY WEIGHTS:
Days 0, 3, 6, 9, 13, 16, 20, 23, 26, 29 post-coitum.

FOOD CONSUMPTION:
Days 0-3, 3-6, 6-9, 9-13, 13-16, 16-20, 20-23, 23-26 and 26-29 post-coitum.

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

POST-MORTEM EXAMINATIONS:
All animals surviving to the end of the observation period, all moribund animals and all animals showing abortion were euthanized and 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). Females which had abortion or spontaneous death, were necropsied within 24 h of abortion.
Ovaries and uterine content:
Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:

- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus.
- Externally visible macroscopic fetal abnormalities.

Animals found dead or sacrificed before planned necropsy, were subjected to relevant examinations of the ovaries and uterine horns.
Fetal examinations:
External, visceral, and skeletal findings were recorded as developmental variations or malformations.

External:
Each viable fetus was examined in detail and weighed. All live fetuses were euthanized. The nonviable fetus (the degree of autolysis was minimal or absent) was examined, crown-rump length measured and weighed. The crown-rump length of late resorptions was measured and a gross external examination performed. 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. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development. 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 for subsequent processing and soft-tissue examination of all groups using the Wilson sectioning technique. After examination, the tissues were stored in 10% formalin. 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 Groups 1 and 4, following fixation in 96% aqueous ethanol, were macerated in potassium hydroxide and stained with Alizarin Red S. Subsequently, the skeletal examination was done on all fetuses from Groups 1 and 4. Since no 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. All specimens 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. 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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITY:
No toxicologically relevant mortalities occurred up to 75 mg/kg bw/day.
Ten animals died preterm
-Five animals showed an abortion and had to be terminated early. Macroscopic examination showed abnormalities of the heart (enlarged, pale or soft), stomach (many black foci on glandular mucosa, many black-brown or dark red foci, irregular surface of the forestomach or gelatinous contents), gallbladder (enlarged or many greenish foci), oviducts (several watery-clear cysts), pancreatic lymph nodes (enlarged and dark red), alopecia, thoracic cavity (containing watery-clear fluid), lungs (many, black foci), liver (pale), general pale discolouration, or watery-clear contents in the caecum.
- Five animals died due to complications of the gavage procedure. Macroscopic examination of these animals revealed abnormalities in the trachea (contents: reddish/dark red foamy or hemorrhagic/clotted blood), lungs (reddish foamy contents, many dark red foci, dark red discolouration or isolated tan focus), or advanced autolysis.

CLINICAL SIGNS
No toxicologically relevant clinical signs were noted up to 75 mg/kg bw/day. Incidental findings that were noted included alopecia, scars, scales, scabs, lean appearance, hunched posture, laboured respiration, rales, ulcer, diarrhoea, a wound, and salivation. These findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS
At 50 and 75 mg/kg bw/day, overall a body weight loss was noted on Day 9 post-coitum and reduced body weight gain was noted on Day 13 post-coitum. These changes were statistically significant but did not show a dose response. During the remainder of treatment, mean body weights recovered to normal. Corrected (for uterus) body weight gain was unaffected at these dose levels. At 25 mg/kg bw/day, body weights and (corrected) body weight gain remained in the same range as controls over the treatment period.


FOOD CONSUMPTION
Absolute and relative food consumption were statistically significantly lower on Day 6-13 post-coitum at 75 mg/kg bw/day and on Day 6-9 post-coitum at 50 mg/kg bw/day. Food consumption was slightly lower on Day 13-16 post coitum (not statistically significant). Subsequently, mean food consumption recovered to normal. Food consumption before or after allowance for body weight of treated animals at 25 mg/kg bw/day remained in the same range as controls.

WATER CONSUMPTION
During the observation period several animals in all groups (vehicle control and test item treated) showed reduced water intake on one or more days. In the absence of a clear treatment-related effect, no quantitative measurement of water consumption was introduced during this study.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence of incidental findings among control and treated animals was within the background range of findings that are encountered among rabbits of this age and strain, and did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

MATERNAL PREGNANCY DATA
For the control, low, mid and high-dose groups, there were respectively 22, 21, 21 and 19 litters with viable fetuses available on Day 29 post-coitum. In the control group, out of 24 mated animals two died preterm; all rabbits were pregnant. At 25 mg/kg bw/day, out of 22 mated animals one had an abortion. At 50 mg/kg bw/day, out of 26 mated animals two aborted, two died preterm (both pregnant) and one was not pregnant. At 75 mg/kg bw/day, out of 24 mated animals two aborted, one died preterm (pregnant) and two were not pregnant.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER SIZE
Litter sizes were not affected by treatment up to 75 mg/kg bw/day. Mean litter sizes were 9.5, 8.6, 9.2 and 8.4 viable fetuses per group for the control, 25, 50 and 75 mg/kg bw/day group, respectively. There was no effect on fetal viability up to 75 mg/kg bw/day. Litter proportions of viable or dead fetuses and early resorptions were all within the normal range of biological variation. The percentage of late resorptions at 25 mg/kg bw/day was slightly outside the historical control data (4.0% versus a maximum of 3.5% in the historical control data). As no dose response was noted, this slight increase was not considered toxicologically relevant.

SEX RATIO
Sex ratio was unaffected by treatment up to 75 mg/kg bw/day.

FETAL BODY WEIGHT
Fetal body weight was unaffected by treatment up to 75 mg/kg bw/day. Mean fetal body weights (both sexes combined) were 36.3, 37.8, 35.5 and 37.5 grams in the control, 25, 50 and 75 mg/kg bw/day group, respectively.

FETAL MORPHOLOGICAL EXAMINATIONS
The numbers of fetuses (litters) available for external and visceral morphological evaluation were 208 (22), 180 (21), 194 (21) and 160 (19) in control, 25, 50 and 75 mg/kg bw/day groups respectively. Soft tissue cephalic examination was done for approximately half of the fetuses of all groups. Skeletal examinations were performed for all fetuses of control and 75 mg/kg bw/day groups.

EXTERNAL MALFORMATIONS AND VARIATIONS
There were no external developmental malformations or variations up to 75 mg/kg bw/day for fetuses at planned necropsy. Two fetuses of one litter from 50 mg/kg bw/day group showed hyperextension of the right hind limb. As this only affected one litter and this malformation was noted in the historical control data, it was not considered toxicologically relevant.

VISCERAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on visceral morphology following treatment up to 75 mg/kg bw/day. Retrocaval ureter was noted at 0.8% in the control group, 1.8% at 25 mg/kg bw/day, 1.1% at 50 mg/kg bw/day and 2.6% at 75 mg/kg bw/day. At 25 and 75 mg/kg bw/day, this was just outside the historical control maximum of 1.4%. Without a clear dose response relationship, these slight increases were not considered toxicologically significant. Any remaining visceral malformations and variations were not considered treatment related as they occurred infrequently, did not follow a dose-related trend, or occurred at frequencies that were within the range of available historical control data.

SKELETAL MALFORMATIONS AND VARIATIONS
At 75 mg/kg bw/day, there was a slight increase in skeletal developmental variations. This included three parameters indicating a developmental delay: unossified metacarpals and/or metatarsals (11.7% at 75 mg/kg bw/day versus 7.7% in the control group), slightly or moderately mal-aligned sternebrae (6.0% at 75 mg/kg bw/day versus 1.8% in the control group) and unossified tarsals (2.9% at 75 mg/kg bw/day versus 1.1% in the control group). As these increases were only slight and not statistically significant, these were not considered toxicologically significant. Additionally, the number of fetuses with caudal shift of the pelvic girdle was increased at 75 mg/kg bw/day (31.7% per litter; 52 fetuses in 11 litters) compared to the control group (17.1% per litter; 36 fetuses in 13 litters). Without any corroborative findings and as the number of affected litters was even less than controls, this finding was not considered toxicologically relevant. All malformations and remaining skeletal variations recorded in this study were not considered to be treatment related as they occurred infrequently, at lower levels in the high dose group, occurred at frequencies that were within the range of available historical control data, or were noted in control fetuses only.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day, since no adverse effect was observed.
Executive summary:

A prenatal developmental toxicity study was conducted with PETIA in rabbits after oral exposure according to OECD Guideline 414, EU method B.31 and EPA OPPTS 870.3700 in compliance with GLP.

Ninety six mated female rabbits were assigned to four dose groups. The number of animals was 24, 22, 26 and 24 for Groups 1, 2, 3 and 4, respectively. The test substance was administered once daily by oral gavage from Days 6 to 28 post-coitum, inclusive, at doses of 25, 50 and 75 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rabbits of the control group received the vehicle, Arachid oil, alone. 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. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations.

No maternal or developmental toxicity was observed in any of the groups. Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity for the test substance was established as being at least 75 mg/kg bw/day, since no adverse effect was observed.