Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 April 2014 - 11 June 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: White powder
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Females were approximately 11 weeks.
- Weight at study initiation: around 219 g average weight
- Housing: Pre-mating: During acclimatization, females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages (MIV type, height 18 cm). Mating: Females were placed in the males’s homecage in Macrolon plastic cages (MIII type, height 18 cm). During the weekend, mating procedures were suspended and the females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages (MIV type, height 18 cm). Post-coitum: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water
- Acclimation period: At least 5 days prior to pairing.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made
for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance. Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
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 (04 June 2014), according to a validated method (Project 505659). 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 (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% 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%.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Details on mating procedure:
Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the female was separated from the male. When sufficient mated females had been obtained for each dose group, the surplus females were removed from the study. During the weekend, mating procedures were suspended.
Duration of treatment / exposure:
From Days 6 to 19 post-coitum, inclusive
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Duration of test:
Day 20 post-coitum
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a range finding study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. The circumstance of any death was recorded in detail.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 sign was 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) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

POST-MORTEM EXAMINATIONS: Yes
All animals were sacrificed on Day 20 post-coitum using an oxygen/carbon dioxide procedure and subsequently 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.
Terminal body weights and liver weights were taken for all animals.
Animals found dead were subjected to relevant examinations of the ovaries and uterine horns.
In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-
Ro water (Millipore Corporation, Bedford, USA)).
The female genital tract including the placentas was preserved in 10% buffered formalin for possible histopathological examination.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
Each viable fetus was examined in detail, sexed and weighed. All live fetuses were euthanized by administration of approximately 0.05 mL (=10mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater) into the oral cavity using a small flexible plastic or metal feeding tube. The
crown-rump length of late resorptions was measured and a gross external examination performed (if possible).

- Soft tissue examinations: Yes: [all per litter]
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 confirmed 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 solution for subsequent processing and soft-tissue examination using the Wilson sectioning technique. After examination, the tissues were stored in 10% buffered formalin. All carcasses, including the carcasses without heads, were eviscerated and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

- Skeletal examinations: Yes: [all per litter of control and high dose]
Each eviscerated fetus of Groups 1 and 4, following fixation in 96% aqueous ethanol, was 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. The skeletal examination was done following this procedure on all fetuses from Groups 1 and 4. Since no possible treatment related effects in the high dose group were seen, skeletal examination were not extended to the fetuses from the low and mid dose group. The specimens were 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 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.

- Head examinations: Yes: [half per litter]
Statistics:
- 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 postimplantation 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:
Pre-implantation loss, Post-implantation loss, Viable fetuses affected/litter

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Details on maternal toxic effects:
MORTALITY
One female at 1000 mg/kg was found dead on Day 13 post coitum. No clinical signs were noted for this female, though she had lost weight (-11%) and had reduced food consumption the days prior to her death. She was noted with beginning autolysis and dark red discoloration of the lungs at the macroscopic examination. Her data are mentioned here, and not included further in the results section. A relationship to treatment could not be excluded.

CLINICAL SIGNS
There were no toxicologically relevant clinical signs noted with treatment up to 1000 mg/kg. Rales were noted for four animals and piloerection was noted for one female at 1000 mg/kg. As these were transient and/or occurred for a limited number of animals, they were not considered to be adverse. Alopecia and scales were incidental findings noted for control and/or high dose females. These findings were not treatment related or toxicologically relevant.

BODY WEIGHT
Body weights and weight gains were significantly lower for females at 1000 mg/kg than controls from Days 9-20 post coitum. Corrected weight gain was also significantly lower for these females. There were no differences in body weights or weight gains between other treated groups and controls.

FOOD CONSUMPTION
Absolute food consumption was lower for females at 1000 mg/kg than controls during the entire treatment period (not always statistically significant). Relative food consumption was significantly lower for high dose females on Days 6-12 only. Relative food consumption recovered thereafter, remaining only slightly lower than controls for the rest of the treatment period (not statistically significant).

MACROSCOPIC EXAMINATION
There were no treatment related macroscopic findings noted up to 1000 mg/kg. A single female at 100 mg/kg had a yellowish focus on the left median lobe of the liver. This was considered incidental. No other macroscopic findings were noted for surviving animals.

ORGAN WEIGHTS
Terminal body weights were lower for females at 1000 mg/kg than controls. There were no differences in liver weights or liver to body weight ratios between control and treated animals up to 1000 mg/kg.

MATERNAL PREGNANCY DATA
There were 20, 19, 21 and 21 pregnant females, and 20, 19, 21 and 20 litters available for evaluation in the control, 100, 300 and 1000 mg/kg groups, respectively. There were no inter-group differences in the number of viable or dead fetuses, early or late resorptions, pre- or post-implantation loss, or in the number of implantation sites or corpora lutea. One female at 1000 mg/kg (no. 86) had twelve early resorptions only. This may have been secondary to maternal toxicity as this female had the lowest food consumption of all Group 4 animals during the initial period after treatment accompanied by body weight loss and low body weight gain.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw (total dose)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
LITTER SIZE
Litter sizes were similar between control and treated animals. Mean litter sizes were 11.2, 10.2, 11.4 and 11.7 fetuses/litter for the control, 100, 300 and 1000 mg/kg groups, respectively.

SEX RATIO
The male:female ratios were similar between control and treated groups.

FETAL BODY WEIGHT
Fetal body weights were unaffected by treatment up to 1000 mg/kg. Mean fetal body weights were 3.4, 3.6, 3.5 and 3.4 grams in the control, 100, 300 and 1000 mg/kg groups, respectively.

FETAL MORPHOLOGIC EXAMINATIONS
The number of fetuses (litters) available for external and visceral morphological evaluation were 223 (20), 194 (19), 239 (21) and 234 (20) in the control, 100, 300 and 1000 mg/kg groups, respectively. Skeletal evaluation was performed for fetuses of the control and high dose groups only and soft cephalic tissue examinations were done for approximately half of the fetuses in each group.

EXTERNAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal external morphology up to 1000 mg/kg. Generalized subcutaneous edema was seen for a single fetus at 300 mg/kg. As only a single fetus was affected and no dose dependent relationship was evident, this was not considered to be treatment related. There were no external variations noted.

VISCERAL EXTERNAL MALFORMATIONS AND VARIATIONS
There were no treatment related effects on fetal visceral morphology seen with treatment up to 1000 mg/kg. Visceral malformations were seen for 1.0, 0.5, 0.7 and 0.8% of fetuses/litter in the control, 100, 300 and 1000 mg/kg groups, respectively. Malformations included situs inversus, abnormal lobation of the lung, absent eye, interrupted aortic arch, atrial septum defect, thoracoschisis and cleft palate. These occurred for individual fetuses, were distributed across the control and treatment groups and/or remained within the available historical control data. Taken together, no malformations were considered to be treatment related or toxicologically relevant. Visceral variations were observed for 9.5, 10.6, 11.1 and 10.4% of the fetuses in the control, 100, 300 and 1000 mg/kg groups, respectively. Visceral variations included liver appendix or small supernumerary lobes, discolored adrenal glands or liver, convoluted or dilated ureter, partially undescended thymus horn and small thyroid. These were not considered attributable to treatment as they occurred at similar frequencies across the groups, occurred infrequently, did not show a doserelated trend and/or occurred at frequencies within the range of available historical control data.

SKELETAL MALFORMATIONS AND VARIATIONS
There were no treatment related or toxicologically relevant effects on fetal skeletal morphology seen up to 1000 mg/kg. Bent limb bones were noted for control and high dose fetuses at a similar frequency (for 5.0 and 3.0%, for control and high dose fetuses/litter, respectively). Supernumerary site on the skull was seen for one fetus at 1000 mg/kg and sternoschisis was seen for two control fetuses. These occurred for a limited number of animals and/or remained within the available historical control data. Skeletal variations were seen for 86.1 and 87.2% of fetuses in the control and high dose group, respectively. These included 14th rudimentary or full ribs, ossification of cervical centrum #1, reduced ossification of the vertebral centra, vertebral arches, or skull, caudal shift of the pelvic girdle, unossified/reduced ossification of the pubis, unossified sternebrae nos. 1,2,3, 4, 5 and/or 6, malaligned sternebrae, branched sternebrae, bent ribs, partially fused zygomatic arch, unossified hyoid, and 7th cervical rudimentary or full ribs. These variations were not considered to be treatment related because they occurred at similar frequencies in the control and 1000 mg/kg group and/or remained within the historical control range.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for the test substance was established at 300 mg/kg and the developmental NOAEL was at least 1000 mg/kg.
Executive summary:

A Prenatal developmental toxicity study was performed with the test article following GLP principles and OECD testing guideline 414. Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered once daily by oral gavage from Days 6 to 19 post-coitum at doses of 100, 300 and 1000 mg/kg (Groups 2, 3 and 4 respectively). The rats of the control group received the vehicle, propylene glycol, alone. 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 20 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 corrected body weights (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 and stained with Alizarin Red S for skeletal examinations. Skeletal examinations were assessed for all fetuses from the control and high dose groups only.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses. At 1000 mg/kg, there was one premature death where a relationship to treatment could not be excluded. For surviving females, maternal toxicity consisted of reduced body weights, body weight gains, body weight corrected for uterine weight and food consumption. No maternal toxicity was observed in the 100 and 300 mg/kg groups. No developmental toxicity was observed in the 100, 300 and 1000 mg/kg groups. Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for the test article was established at 300 mg/kg and the developmental NOAEL was at least 1000 mg/kg.