2-Propenamide, N-[4-[[(4,6-dimethyl-2-pyrimidinyl)amino]sulfonyl]phenyl]-2-methyl-

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

28 days exposure

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Qualifier:

equivalent or similar to guideline

Guideline:

other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)

Version / remarks:

2008

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
This type of study plan was reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee of Charles River Den Bosch as required by the Dutch Act on Animal Experimentation (February 1997).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age/weight at study initiation: At initiation of dosing, males were 10 weeks old and weighed between 271 and 308 g and females were 13 weeks old and weighed between 210 and 249 g.
- Fasting period before study: no
- Housing:
On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage- enrichment, bedding material, food and water.
The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.
- Diet (e.g. ad libitum):
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water (e.g. ad libitum):
Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period:
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pre-test period (females) or 7 days before the commencement of dosing (males).

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility.
It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: dosing was initiated on 03 May 2017. To: The in-life phase of the study was completed on 05 Jul 2017.

Route of administration:

oral: gavage

Details on route of administration:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days.
The oral route of administration was selected because this is the most likely route of human exposure.

Vehicle:

carbowaxe

Remarks:

1% Aqueous carboxymethyl cellulose

Details on oral 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 as a suspension and dosed within 5 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. No adjustment was made for specific gravity of the vehicle and test item or for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after completion of the assessment. These trial preparations have a non-GLP status and were carried out in the quality assured environment of the Test Facility.
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): Documented in raw data.

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. 17054).

Dose formulation samples were collected for analysis as indicated below.
Dose Formulation Sample Collection Schedule: All groups were samples on day 2 of treatment. Homogeneity was checked for groups 2 and 4.

All samples were stored on dry ice immediately after sampling. All samples to be analyzed were shipped on dry ice to ABL B.V. on the day of sampling. The analytical laboratory was notified before shipment of the samples. Upon receipt at the analytical laboratory, the samples were stored in the ultra-low freezer ≤ -70 °C until analysis.

ConcentrationAnalysis
Duplicate middle samples for Groups 1 and 3 and duplicate top, middle, and bottom samples for Groups 2 and 4 (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% for suspensions of target concentration.
Homogeneity Analysis
Duplicate top, middle, and bottom samples for Groups 2 and 4 (approximately 500 mg accurately weighed) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (ABL No. 17053) 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 ABL No. ABL17053. In this study, stability for at least 5 hours at room temperature, 8 days in the refrigerator and 3 weeks in the freezer was confirmed over the concentration range 1 to 200 mg/mL.

Duration of treatment / exposure:

Males were treated for 29 days, up to and including the day before scheduled necropsy, i.e. two weeks prior to mating and during the mating period. Females were treated for 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy (females with offspring: 50-55 days (most females) or 62-63 days (two females); females without offspring: 42 or 51 days).

Frequency of treatment:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

all doses were administered to 10 males and 10 females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were selected based on the results of a 10-day oral dose range finder with V123109 in rats (Test Facility Study No. 517102), and in an attempt to produce graded responses to the test item.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Mortality
F0: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
F1: Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter.
Clinical observations
F0: Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
The time of onset, grade and duration of any observed sign was recorded.
F1: Clinical observations were performed at least once daily for all pups.
Cohabitation/mating
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. 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 males and females were separated.
A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
General reproduction data
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day.

DETAILED CLINICAL OBSERVATIONS: Yes
The animals were removed from the cage and placed in a standard arena, and a detailed clinical observation was performed weekly, beginning before the first administration of the test item. These observations were conducted during the dosing interval.

BODY WEIGHT: Yes
F0: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
A fasted weight was recorded on the day of necropsy.
F1: Live pups were weighed individually on PND 1, 4, 7 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:
/

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
Blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. After collection, all samples were transferred to the appropriate laboratory for analysis.
Blood of F1-animals was collected on PND 4 and PND 13-15.
On PND 4 at culling, blood was collected from two surplus pups per litter by decapitation, between 7.00 and 10.30 a.m., and samples were pooled to one sample per litter. If available, blood was collected from one male and one female pup.
On PND 13-15, separate blood samples were collected from two pups per litter (from one male and one female, if possible). Blood was drawn between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) as part of the necropsy procedure.
- Animals fasted: Yes: F0-animals were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
- How many animals: 5 per treatment group
- Parameters checked; see Table 1


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: See Haematology
- Animals fasted: See Haematology
- How many animals: 5 per treatment group
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. PND 6-13). These tests were performed after completion of clinical observations (including arena observation, if applicable).
- Dose groups that were examined: all treatlent groups
- Battery of functions tested:
sensory activity: hearing ability, pupillary reflex and Static righting reflex
grip strength: Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal
motor activity: Locomotor activity. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

OTHER:
ESTROUS CYCLE DETERMINATION
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pre- test period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females
BLOOD COAGULATION
Blood samples were processed for plasma, and plasma was analyzed for the parameters Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)
THYROID HORMONE
Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)

HISTOPATHOLOGY: Yes (see table 3)

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.

The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

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 was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis.
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 Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or
during weekly arena observations.
One female at 1000 mg/kg (no. 71) showed abdominal swelling starting after 5 weeks of treatment. This correlated with the nodule observed at necropsy which was diagnosed as a mammary gland adenocarcinoma and was not regarded as treatment-related. This female delivered a normal litter size.
All other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment
with the test item.
One control male (no. 7) was sacrificed on Day 2 of treatment since this animal was reported to have swallowed a significant part of the gavage tube. This animal was subsequently replaced by a reserve animal on the same day. No histopathology was conducted for the original animal no. 7.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight and body weight gain were not considered to be affected by treatment.
The statistically significantly higher mean body weight noted at 100 mg/kg in females on Day 7 of lactation was considered unrelated to treatment due to the lack of a dose-related trend.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related changes in food consumption before or after allowance for body weight
were noted.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in haematological parameters (red and white blood
cell parameters, number of platelets).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg, a statistically significantly higher mean fasting glucose value (30% higher than control means) was noted in female rats. The mean glucose value in 1000 mg/kg females remained within the historical control range.
Other statistically significant variations noted in clinical biochemistry parameters were considered unrelated to treatment due to the lack of a dose-related trend (higher sodium at all dose levels in males) or direction of the change (lower bilirubin at 1000 mg/kg in both sexes).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not considered affected by treatment; the statistically significantly lower mean hind limb grip strength values in all groups of treated females were not considered related to treatment since a clear dose-related response was absent, forelimb grip strength was similar to the control mean and since means remained within the range considered normal for rats of this age and strain1.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations.
There was one female treated at 1000 mg/kg (no. 71) with a mammary gland adenocarcinoma. This correlated with the black, yellowish hard, nodule of 18x15 mm observed at necropsy. Although mammary gland adenocarcinoma is a rare finding at this age, mammary gland lesions are one of the most commonly occurring natural neoplastic lesions in this rat strain. Moreover, this specific type is the most commonly observed type in the age range of 6 to 38 weeks (Barsoum et al.). In the absence of any morphological lesions in the mammary gland of other animals, this finding was not regarded as treatment-related and its occurrence in a single high dose female was considered to be a chance finding.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in coagulation parameters (prothrombin time and activated partial prothrombin time).
Serum levels of T4 in F0 males were considered not to be affected by treatment.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

neuropathology

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of V123109 were established:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg

Executive summary:

The objectives of this study were to determine the potential toxic effects of V123109 when given orally by gavage for a minimum of 28 days to Wistar rats and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg/day, based on the results of a dose range finder.

The study design was as follows:

Text Table 1 Experimental Design

Group No.	Test Item Identification	Dose Level (mg/kg/day)	Dose Volume (mL/kg)	Dose Concentration (mg/mL)	Number of Animals
					Males	Females
1	-	0 (Vehicle)1	5	0	10	10
2	V123109	100	5	20	10	10
3	V123109	300	5	60	10	10
4	V123109	1000	5	200	10	10

11% Aqueous carboxymethyl cellulose.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following endpoints were evaluated in this study: mortality/ moribundity, clinical signs, functional observations and locomotor activity (5 selected rats/sex/group), body weight and food consumption, estrous cycle length and regularity, clinical pathology (5 selected rats/sex/group), serum level of thyroid hormone T4 (F0-males and PND 13-15 pups), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention and macroscopy).

Accuracy and homogeneity of dosing formulations were confirmed by analyses.

No parental, reproduction or developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

A higher mean plasma level of fasting glucose noted in 1000 mg/kg females (1.3x of control) was regarded as non-adverse as this change was not associated with adverse anatomic pathology findings and glucose values in treated females remained within normal limits.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of V123109 were established:

Parental NOAEL:

at least 1000 mg/kg

Reproduction NOAEL:

at least 1000 mg/kg

Developmental NOAEL:

at least 1000 mg/kg

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat