Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Aug 2020 - 22 Dec 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was initiated after final decision CCH-D-2114484966-27-01/F with the request to submit a Pre-natal developmental toxicity study (Annex IX, Section 8.7.2.; test method: OECD TG 414) in a first species (rat or rabbit), oral route with the registered substance was received.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
432-520-2
EC Name:
-
Cas Number:
232938-43-1
Molecular formula:
C21 H20 N2 O6 S2
IUPAC Name:
3-{[(4-methylbenzenesulfonyl)carbamoyl]amino}phenyl 4-methylbenzene-1-sulfonate
Test material form:
solid: particulate/powder
Details on test material:
Physical Description: Off-white powder
Specific details on test material used for the study:
Storage conditions: At room temperature protected from light
Test item handling: No specific handling conditions required
Purity/Composition correction factor: No correction factor required (water content: 0.080%)
Volatile: No
pH: 6.82 at a concentration of 10% in distilled water

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks (Day 0 or Day 1 post coitum (Day post coitum: day of successful mating))
- Weight at initiation of dosing: 189 - 263 g
- Fasting period before study: No
- Housing: Individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Municipal tap water, ad libitum (available via water bottles)
- Acclimation period: At least 5 days

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed. No known contaminants were present in the feed or water that would interfere with the objectives of the study. For psychological/environmental enrichment and nesting material, animals were provided with paper and aspen wooden sticks, except when interrupted by study procedures/activities. It was considered that there were no known contaminants that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 45-69
- Air changes (per hr): 10 or more (fresh air, no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 October 2020 To: 13 November 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
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. Prior to use, an appropriate amount of test item was ground using a mortar and pestle. Subsequently, formulations of test item in vehicle were prepared using a mortar and pestle after which formulations were stirred until homogeneous. The dosing formulations were prepared daily as a suspension and dosed within 4 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: 0, 4, 12 and 40 mg/mL for the control, low, mid and high dose group formulations, respectively
- Amount of vehicle: 5 mL/kg bw
- Stability for at least 4 hours at room temperature under normal laboratory light conditions for 1 mg/mL formulations, for at least 24 hours at room temperature under normal laboratory light conditions for 200 mg/mL formulations, for at least 8 days in the refrigerator for 200 mg/mL formulations and for at least 3 weeks in the freezer (≤-15°C) for 0.5 mL samples was confirmed over the concentration range 1 to 200 mg/mL (suspensions), Charles River Laboratories Den Bosch Project: 20248633
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis in week one for concentration determination (all groups) and homogeneity determination (low and high dose group). Analyses were performed using a validated analytical procedure (Charles River Laboratory Den Bosch Study No. 20248633).
Concentration results (duplicate sets of samples were measured) were considered acceptable if mean sample concentration results were within or equal to ±15% for suspensions of target concentration. Homogeneity results (also measured in duplicate samples) were considered acceptable if the coefficient of variation (CV) of concentrations was ≤10%.
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20248633) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations used in the present study.
Details on mating procedure:
Time-mated female Wistar Han Rats were received from the breeder. The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
Duration of treatment / exposure:
15 days (Day 6 to Day 20 post-coitum, inclusive)
Frequency of treatment:
Once daily, 7 days a week
Duration of test:
16 days
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
22
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of the dose range finder. No guidelines were applicable as this study was intended for dose level selection purposes only. If not mentioned otherwise, test system, procedures and techniques were identical to those used during the main study. The test item and vehicle were administered to the appropriate animals (6 females/ dose group) by once daily oral gavage from Day 6 to Day 20 post-coitum, inclusive. The dose levels were selected based on the outcome of the screening study (as summarized in this dossier). Six females per dose were treated at 0, 100, 200 and 375 mg/kg bw/day. In-life Procedures, observations, and measurements in the DRF were identical as for the main study, except for anogenital distance measurements and blood collection for thyroid hormone analysis, which were not conducted. Terminal procedures of F0-animals were identical as for the main study, except for thyroid weight measurements and fixation of the thyroid, which were not conducted. Each viable fetus of animals surviving to planned necropsy was externally examined in detail, weighed and sexed.
All live fetuses were euthanized by decapitation. For late resorptions, a gross external examination was performed. Fetuses of animals found dead or sacrificed before planned necropsy were externally examined in detail and euthanized by decapitation (if necessary). No visceral (internal) or skeletal examination was performed. The results of this DRF are given below.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Cage debris was examined to detect premature birth.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly, beginning during the Pre-Treatment Period, and on the day of necropsy.
- The animals were removed from the cage, and a detailed clinical observation was performed


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum

FOOD CONSUMPTION: Yes
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and
18-21 post-coitum

WATER CONSUMPTION: Yes
- Water consumption was monitored on regular basis throughout the study by visual inspection
of the water bottles/containers

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- All animals 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). The liver, kidneys, gravid uterus and thyroid gland were weighed. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated.
- Thyroid glands of all animals were examined histopathologically.
Ovaries and uterine content:
Each ovary and uterine horn of all animals were dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the uterus.
- The number of implantation sites.
- The number and distribution of live and dead fetuses.
- The number and distribution of early and late resorptions.
- The sex of each fetus based on the anogenital distance, if possible.
In case no macroscopically visible implantation sites were present, nongravid uteri (in this study 3 control females, and 1 female dosed at 20 mg/kg bw/day) were stained using the Salewski technique in order to detect any former implantation sites.
Blood sampling:
Blood of F0-animals was collected on the day of scheduled necropsy. Animals were not fasted overnight. Samples were collected randomized between 7:00 and 9:00 a.m. from the jugular vein.
Thyroid Hormone Parameters (Triiodothyronine (T3), Thyroxine (T4) and Thyroid-stimulating hormone (TSH)) were measured.
Fetal examinations:
Fetuses were euthanized by administration of sodium pentobarbital (oral cavity).
- Each viable fetus was sexed, examined in detail to detect macroscopic visible abnormalities
and their weight was determined. The anogenital distance (AGD) was measured for all viable fetuses. The AGD was normalized to the cube root of the fetal body weight. For late resorptions (41-L6, 70-L5), a gross external examination was performed.
- The sex of all fetuses was confirmed by internal examination and approximately one-half of
the fetuses (live and dead) in each litter (all groups) 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 heads were removed from this one-half of the fetuses in each litter and placed in Bouin's solution for soft-tissue examination using the Wilson sectioning technique. After examination, the tissues without variation or malformations were discarded. Tissues with variations or malformations (1-L2) were stored in 10% formalin.
- All fetuses were eviscerated, followed by fixation in 96% aqueous ethanol, and maceration in
potassium hydroxide. Thereafter, they were stained with Alizarin Red S by a method similar
to that described by Dawson. Subsequently, skeletal examination was done for one-half of the fetuses (i.e. the fetuses with heads).
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% or 5% levels. The following pairwise comparisons were made: Low dose group vs. control group, mid dose group vs. control group, high dose group vs. control group. Analyses were performed according to the matrix below (under Any other information on materials and methods incl. tables) when possible but exclude any group with less than 3 observations.
Levene’s test (parametric/non-parametric values) was used to assess the homogeneity of group variances. The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. The groups of non-parametric values (ovarian and uterine examinations, litter % of fetuses with gross/external/visceral/skeletal abnormalities) were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found significant, then the above pairwise comparisons were conducted using Dunn’s test. The data corresponding to a response variable of interest and to a related covariate were submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett’s test. A Fisher’s exact test was used to conduct pairwise group comparisons of interest (incidences).

The variables are summarized under "Indices".
Indices:
Maternal Variables:
Body Weight Gains: Calculated for the following intervals: Days 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, and 6 to 21 post-coitum.
Corrected Body Weight Gain: Body weight on Day 21 post-coitum - body weight on Day 6 post-coitum - gravid uterus weight.
Overall Food Consumption: Calculated between each scheduled interval (individual data only) and as specified above for bodyweight gains. Summarization and statistical analysis intervals will reflect the same intervals as the bodyweight gains.
Pregnancy Rate (%): (No. of pregnant females/ No. of mated females) x 100

Litter Variables
Sex Ratio (% males): (No. male fetuses/ Total no. of fetuses) x 100
Pre-Implantation Loss (%): ((No. of corpora lutea – no. of implants)/ No. of corpora lutea) x 100
Post-Implantation Loss (%): ((No. of implants – no. of live fetuses)/ No. of implants) x100
Litter % of Fetuses with Abnormalities: (No. of fetuses in litter with a given finding/ No. of fetuses in litter examined) x100
Historical control data:
Historical control ranges were included where relevant.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Erected fur was noted at 200 mg/kg bw/day in 6/22 animals on 6-11 days, mainly towards the end of the study. At 60 mg/kg bw/day, it was noted in 3/22 animals on 2-5 days towards the end of the study. Erected fur noted in a single animal at 20 mg/kg bw/day from Day 16 post-coitum onwards was considered not toxicologically relevant given the occurrence in a single animal. Any other clinical signs noted, i.e. fur loss and skin scabs of the dorsal cervical area, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day, body weight and body weight gain were decreased on most occasions. No
body weight gain was noted between Days 6-9 post-coitum. In this period, a body weight loss
down to 0.92x compared to Day 6 was noted in 9/22 individual animals. From Days 9-15
post-coitum, body weight gain was comparable to control. From Day 15 post-coitum onwards, body weight gain was decreased again, resulting in a decreased terminal body weight (0.91x of control). Corrected body weight gain for gravid uterus weight of dams at 200 mg/kg bw/day was also decreased (0.58x of control). At 20 and 60 mg/kg bw/day, no effects on body weight or (corrected) body weight gain were observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg bw/day, food consumption was reduced throughout the treatment period (down to
0.76x of control between Days 6-9 post-coitum, and overall to 0.85x between Days 6-21 post-coitum). At 60 mg/kg bw/day, a reduction in food consumption was noted between Days 6-9 post coitum (0.90x of control) with recovery to control levels by the end of the treatment period.
Food consumption at 20 mg/kg bw/day was considered to be unaffected by treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean total Triiodothyronine (T3) levels were reduced at 60 and 200 mg/kg bw/day (0.85x and
0.64x of control mean, respectively). However, as mean levels remained within the available
historical control range (see below), the observed reduction was considered not toxicologically relevant. Mean total Thyroxine (T4) levels were reduced at 60 and 200 mg/kg bw/day (0.87x and 0.57x of control mean, respectively). At 60 mg/kg bw/day, given the small magnitude of the effect and as mean T4 levels remained within the available historical control range (see below), the observed decrease was considered not toxicologically relevant. At 200 mg/kg bw/day, however, the mean T4 level (and individual serum levels of 17/22 females) were below the lower limit of the available historical control range (given below). Based on the magnitude of the effect and as T4 values in the high dose group were below the historical control range, a relation to treatment with the test item could not be excluded.
Although not statistically significant, mean serum levels of thyroid stimulating hormone (TSH) showed a similar trend towards a decrease at 60 and 200 mg/kg bw/day (0.85x and 0.77x
of control, respectively). This apparent decrease may have arisen as a result of slightly high
control values (given below) in combination with individual animals with low levels of TSH compared to group average. As all mean values remained well within the available historical control range (given below) the observed decrease in TSH was considered not toxicologically relevant.

Historical control data for pregnant Wistar Han rats (period 2020)
T3 (ng/mL) mean: 0.437, P5-P95: 0.280 – 0.603 (n = 197)
T4 (ng/mL) mean: 23.3, P5-P95: 16.0 – 35.6 (n = 88)

Historical control data for pregnant Wistar Han rats (period 2016-2020)
TSH (mU/L) mean: 0.353, P5-P95: 0.127 – 0.699 (n = 373)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver:body weight ratios were increased for animals treated at 60 and at 200 mg/kg bw/day
(1.06x and 1.15x of control, respectively). This increase was considered secondary to the
decreased terminal body weights (although not statistically significantly decreased at 60 mg/kg bw/day) and given the small magnitude of the response considered not toxicologically
relevant. There were no test item-related alterations in thyroid gland or kidney weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related gross observations. Findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations. The recorded microscopic findings
were within the range of background pathology encountered in rats of this age and strain.
There was one finding of note in the thyroid gland of one female dosed at 200 mg/kg bw/day, that showed microscopic unilateral a moderate granulomatous inflammatory process, which correlated to macroscopic enlargement of the right thyroid gland. This is an unusual alteration, but this unilateral inflammatory lesion in a single female was regarded incidental and unrelated to the treatment with the test item.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The pre-implantation loss in the control and treated groups was similar and in the range of normal biological variation (mean pre-implantation loss of 11.22, 13.82, 9.97 and 4.44% for the control, low, mid and high dose group, respectively). The mean post-implantation loss was 2.96, 3.66, 2.00 and 5.48% for the control, low, mid and high dose group, respectively. The apparent increase in post-implantation loss at 200 mg/kg bw/day (although not statistically significant) was considered to have arisen as a result of low control values compared to historical control data and regarded to be of no toxicological significance.

Historical control data for pregnant female Wistar Han rats (period 2016-2020):
Post-implantation loss (%/litter): mean: 5.2; P5-P95: 2.1 – 9.0 (n=1317).
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of early resorptions was 0.4, 0.4, 0.2 and 0.6 for the control, low, mid and high dose groups, respectively. The mean number of late resorptions was 0.0 for all groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
No dead fetuses were found in any of the groups.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
At scheduled necropsy, three control females and one female doses at 20 mg/kg bw/day were
nongravid. All pregnant females had live fetuses. Therefore, the number of females with viable litters for evaluation was 19, 21, 22, and 22 in the control, 20, 60 and 200 mg/kg bw/day
group, respectively.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetal body weights at 200 mg/kg bw/day were decreased: 0.91x, 0.88x and 0.90x of control
for male, female and combined fetal body weights, respectively. Fetal body weights at 20 and 60 mg/kg bw/day were considered unaffected by treatment with the test item.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 200 mg/kg bw/day.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no test item-related effects on fetal anogenital distance (both sexes) noted after
treatment up to 200 mg/kg bw/day.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External malformations were observed in one fetus each at 60 and 200 mg/kg bw/day and in two
control fetuses. The high dose fetus had cleft palate with an underlying skeletal abnormality and the mid dose fetus had edema of the entire subcutis. As these external malformations occurred singly, they were considered not test item-related. Moreover, one of the control fetuses had no lower jaw and a cleft lip and also appeared to have cleft palate at fixed head examination. The other affected control fetus was considered completely malformed due to presence of several external and skeletal abnormalities. External variations were not noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were noted in 2 (2), 2 (2), 5 (4) and 3 (3) fetuses (litters) of the control, 20, 60 and 200 mg/kg bw/day groups, respectively. Bending of limb bones (especially the humerus) occurred most frequently. This malformation was observed in four fetuses (from three different litters) in the mid-dose group and in one fetus each of the control and low-dose group. However, a relation to the test item could not be established as no cases occurred in the high dose group.
Two other malformations (multiple skull malformations in one control fetus and split palatine bones in a high dose fetus), were related to an external malformation and the remaining ones occurred singly. Therefore, these were not considered related to treatment with the test item.
Among variations, there were no signs of delays of ossification that were associated with the
lower fetal weights at 200 mg/kg bw/day. On the contrary, incomplete ossification of the
interparietal and both parietal skull bones occurred at lower incidences at the high dose than
in other groups. The reason for this is unknown and a relation to the test item is unclear, however, this is regarded not an adverse effect.
Remaining skeletal variations occurred in the absence of a dose-related trend and/or infrequently. Therefore, they were considered to be not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral malformations were observed in one fetus each at 60 and 200 mg/kg bw/day and in
two control fetuses. With the exception of situs inversus that occurred in a mid-dose fetus
(general) and a control fetus (thoracic), there was no resemblance in any respect between the
observed abnormalities. Therefore, all were considered chance findings.
Visceral variations that have occurred more than once only affected the liver (supernumerary
lobes) and for the low incidences and group distribution this finding did not indicate any test
item-relationship.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Any other information on results incl. tables

Dose Range Finding Study


Maternal findings: At 375 mg/kg bw/day, three animals did not survive until scheduled necropsy, two of these mortalities were considered related to treatment with the test item. One female was found dead in its cage prior to dosing on Day 8 post-coitum. On this day, it was noted with hunched posture and piloerection. During the pre-treatment period, normal body weight (gain) and food consumption were noted. Macroscopic findings comprised of a diaphragmatic hernia in the right medial lobe of the liver and a dark black multifocal glandular focus in the stomach. One high dose female was euthanized for animal welfare reasons on Day 18 post-coitum. During the last week, it presented with hunched posture, erected fur, pallor skin and yellow or pale faeces. Food consumption was very low since the start of dosing and its body weight had barely increased. At necropsy, this female was pregnant with viable fetuses. No macroscopic abnormalities were noted. The third high dose female had an early delivery on the day of scheduled necropsy (1 dead fetus, 5 viable fetuses). It was noted with erected fur and hunched posture from Day 10 and 12 post-coitum onwards, respectively. Yellow and pale feces and pallor skin were noted on a few days. In addition, abnormal breathing was noted on a single day (Day 13 post-coitum). No effects on body weight or food consumption and macroscopic abnormalities were noted. Clinical signs in the remaining animals at 375 mg/kg bw/day consisted of hunched posture and erected fur from Day 9 or 10 post-coitum onwards. In one animal, yellow feces on Day 13-14 post-coitum, and pale skin from Day 14 post-coitum onwards were also noted. At 200 mg/kg bw/day, erected fur was noted in 3/6 animals between Days 10-14 post-coitum. Salivation after dosing (up to a moderate degree) was noted in animals of all treated groups on multiple consecutive days throughout the treatment period from Day 12 post-coitum. This sign was considered to be a physiological response rather than a sign of systemic toxicity. At 375 mg/kg bw/day a slight mean body weight loss (6%) was noted between Days 6-9 post-coitum. Moreover, mean body weight remained lower than concurrent control throughout the treatment period (0.67x of control on Day 21 post-coitum, n=3). The decreased body weight at Day 21 post-coitum was partly due to the absence of viable fetuses at scheduled necropsy (also see Fetal findings below). Body weight gain corrected for gravid uterus weight was reduced (0.16x of control, n=3).
At 200 mg/kg bw/day, a similar trend was observed with no mean body weight gain between Days 6-9 post-coitum (slight body weight loss in 3/6 animals, up to 5% in individual animals). At the end of the treatment period, body weight (gain) at 200 mg/kg bw/day was lower compared to concurrent controls (up to 0.94x for mean body weights). Body weight gain corrected for gravid uterus weight was reduced (0.41x of control). Body weights and body weight gains were considered unaffected at 100 mg/kg bw/day.
Lower mean food consumption at 375 mg/kg bw/day was noted from Day 6 post-coitum onwards (down to 0.53x of control between Days 15-18 post-coitum, n=5). A similar trend was noted at 200 mg/kg/day (down to 0.72x of control between Days 6-9 post-coitum). At 100 mg/kg bw/day, food consumption was considered unaffected. The kidney: body weight ratios were significantly increased at 375 mg/kg bw/day (1.45x of control). Liver: body weight ratios were increased: 1.14x, 1.22x and 1.30x of control at 100, 200 and 375 mg/kg bw/day, respectively. Macroscopic observations at necropsy revealed a diaphragmatic hernia in the right medial lobe of the liver of one of the females at 200 mg/kg bw/day. Of note: this finding was also noted in the female at 375 mg/kg bw/day) that was found dead on Day 8 post-coitum, but given the nature of this finding, it was considered not toxicologically relevant.
Pale fluid accumulation in the uterus of one of the females dosed at 375 mg/kg bw/day is likely related to a stage in the estrous cycle and was considered a normal finding. At scheduled necropsy, there were no females with viable fetuses at 375 mg/kg bw/day. Salewski staining of the uterus of the three surviving females showed several empty implantation sites, indicating that the pregnancies were lost early on during gestation. For these animals, the postimplantation loss was 100%. Moreover, the observed increased pre-implantation loss at 375 mg/kg bw/day might also be related to this, as the loss of implantations very early in the pregnancy cannot be visualized at scheduled necropsy on Day 21.
Fetal findings: Mean fetal body weights at 200 mg/kg bw/day were decreased: 0.87x, 0.89x and 0.89x of control for male, female, and combined fetal body weights, respectively. The number of late resorptions, pre-implantation loss and sex ratio were considered unaffected by treatment up to 200 mg/kg bw/day. No external malformation were noted.


Based on the results of the DRF, selected dose levels for the main study were 20, 60 and 200 mg/kg bw/day.


Formulation analyses


No test item was detected in the control group formulation. The mean accuracy of the mid dose group was slightly below target (i.e. 82%). An out of specification investigation was initiated to find the cause of this deviation from target. No clear cause was found in formulation preparation or analytical procedures. However, during the trial formulation analysis in Charles River Laboratories Den Bosch Study No. 20248633, it was observed that formulations and pretreated samples have limited stability since the test item reacts with propylene glycol. The concentrations analyzed in the mid and high dose group formulations were in agreement with target concentrations (i.e. mean accuracies between 86% and 89%).

The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation ≤ 3.3%).

Applicant's summary and conclusion

Conclusions:
Based on the results of a prenatal study in rats with exposure via oral gavage, the maternal and developmental NOAEL for Pergafast 201 were established at 60 mg/kg bw/day (based on reduced body weight gain and food intake and decreased fetal body weights at 200 mg/kg bw/day).
Executive summary:

A prenatal study in rats with exposure via oral gavage was performed with Pergafast 201 at the dose levels 0, 20, 60 and 200 mg/kg bw/day. The dose levels were determined based on the results of a dose range finder study. The study was performed according to OECD/EC guidelines and GLP principles. Time-mated female Wistar Han rats were exposed from Day 6 to 20 post-coitum, inclusive. Test formulation accuracy was considered acceptable and formulations were homogeneous at the concentrations tested. No mortality occurred during the treatment period. At 200 mg/kg bw/day, erected fur was recorded for several animals mainly towards the end of the study. Body weight and body weight gain were decreased on most occasions, resulting in a decreased terminal body weight. Corrected body weight gain for gravid uterus weight was also decreased. This was accompanied by a lower mean food consumption throughout the treatment period. No maternal toxicity was observed at 20 and 60 mg/kg/day. In this study, a reduction of Total T4 was observed in the high dose group which was considered to be test item-related. There were no test item-related microscopic observations. As possible adversity of the effect on Total T4 could not be assessed within this type of study it was not taken into account when determining the maternal NOAEL.


Mean fetal body weights were decreased for male and female fetuses at 200 mg/kg bw/day compared to concurrent controls, as well as combined fetal body weights. It cannot be ruled out that this decrease was secondary to the observed maternal toxicity at 200 mg/kg bw/day. Based on the magnitude of the effect (approximately 10%), this was considered to be test item-related and adverse. No developmental toxicity was observed at 20 and 60 mg/kg/day. Based on these results, the maternal and developmental NOAEL for Pergafast 201 were concluded to be 60 mg/kg bw/day.