Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined repeated dose toxicity study with reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only 7 males in control/high dose groups; no historical lab data provided.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

EC Number:

950-492-4

Cas Number:

2361378-62-1

Molecular formula:

C21-23H39-43NO3

IUPAC Name:

Reaction mass of 2- (palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate

impurity 1

Reference substance name:

Amides, C16-18, N-(hydroxyethyl)

EC Number:

309-819-0

EC Name:

Amides, C16-18, N-(hydroxyethyl)

Cas Number:

101226-97-5

IUPAC Name:

Amides, C16-18, N-(hydroxyethyl)

impurity 2

Reference substance name:

Zirconium compounds

IUPAC Name:

Zirconium compounds

Test material form:

solid: granular

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Provided by sponsor, lot number: 91112Y
- Purity: 92.4%.


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (actual temperature: 18.0°C to 23.8°C; permissible range: 1°C to 30°C), in a dark place, in tight containers, Test Substance Storage Room A046


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Dosing formulations for the test substance groups were stirred with a magnetic stirrer during administration.


Purity correction was applied.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc. (Hino Breeding Center)
- Age at study initiation: 9 weeks old
- Weight at study initiation: Males: 324.8 to 382.8 g (permissible range: 286.8 to 430.2 g); Females: 190.0 to 253.8 g (permissible range: 184.7 to 277.1 g)
- Fasting period before study: Animals were only fasted before scheduled necropsy for 16 hours or more
- Housing: (1) For males and females except during gestation and lactation periods
Hanging type stainless wire mesh cages with a resting board, nesting materials (HAPPIMATS [Marshall BioResources] and expanded Diamond Twists [ENVIGO]), and a gnawing material (Diamond Twists). Exchange: at least once every 14 days (7- to 14-day interval)

(2) For females during gestation and lactation periods
Polymethylpentene cages with nesting material (Paper Clean [Japan SLC, Inc.]) and a gnawing material (Diamond Twists). Exchange: at least once a week but no exchange from Day 21 of gestation to Day 3 of lactation (3- to 7-day interval)

2 or 3 animals per cage before grouping; 1 male and 1 female per cage during the mating period, 1 dam and its litter per cage during the lactation period, and 1 animal per cage for the other periods after grouping.

- Diet: Radiation-sterilized pellet diet (CRF-1, Oriental Yeast Co., Ltd.) ad libitum
- Water: Well water admixed with NaClO (free residual chlorine level: about 0.2 ppm) ad libitum
- Acclimation period:14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.4°C to 24.7°C
- Humidity (%):25.6% to 60.4%
- Air changes (per hr):10 to 20 times per hour
- Photoperiod (hrs dark / hrs light):12 hours per day (7:00 to 19:00)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test substance was administered orally using a disposable syringe attached to a gastric
tube. Dosing formulations for the test substance groups were stirred with a magnetic
stirrer during administration.

Vehicle:

other: 0.5 w/v% methylcellulose (MC) solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing formulations were prepared at least once every 11 days (actual frequency: 6- to 8- day interval) based on the results of the analytical method validation for the determination, and homogeneity and stability test (study No. P200500) conducted at the test facility. The dosing formulations after preparation were divided into glass vials for each dosing day and stored in a cold place (actual temperature: 3.3°C to 6.2°C, permissible range: 1°C to 15°C, storage area: a medical refrigerator) in which they were confirmed to remain stable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5 w/v% Methylcellulose Solution
The vehicle was selected based on the results of a study entitled “Analytical Method Validation for the Determination, and Homogeneity and Stability Test of Reaction mass of 2-(palmitoylamino)ethyl acrylate and 2-(stearoylamino)ethyl acrylate in 0.5 w/v% Methylcellulose Solution (Study No. P200500)” conducted at the test facility.
Methylcellulose 400; Manufacturer: FUJIFILM Wako Pure Chemical Corporation; Lot number: CAM6671
Water for injection, Japanese Pharmacopoeia; Manufacturer: Otsuka Pharmaceutical Factory, Inc.; Lot number: 8K83

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At the first preparation, 10 mL each of the analytical samples were taken from one point
of each layer (upper, middle, and lower layers, n=1 in each layer) of the whole dosing
formulation at each concentration. The test substance concentration in the dosing
formulations was analyzed according to the method validated in the analytical method
validation for the determination, and homogeneity and stability test (study No. P200500)
conducted at the test facility.

The relative standard deviation (RSD) of analytical values (concentration) in each layer
should not be more than 10%, and measured concentration (mean) should be within
100±10% as the ratio to the nominal concentration.

Duration of treatment / exposure:

1. Males: From 14 days before mating until day before the necropsy throughout the mating period (42 days in total)

2. Females: From 14 days before mating until Day 13 of lactation (day of delivery was designated as Day 0 of lactation) throughout the mating and gestation periods and delivery. Non-copulated females were kept until day before the necropsy.

3. Recovery/satellite males/females: For 42 days without mating (the same period with that for males)

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

330 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

110 mg/kg bw/day (nominal)

Doses / concentrations 4

No. of animals per sex per dose:

Dosing period
0 mg/kg bw/day: male 7; female: 12
110 mg/kg bw/day: male 12; female: 12
330 mg/kg bw/day: male 12; female: 12
1000 mg/kg bw/day: male 7; female: 12

Recovery period
0 mg/kg bw/day: male 5; female: 5
1000 mg/kg bw/day: male 5; female: 5

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on a 14 day DRF (Supporting, RL2, DRF/LSIM, 2021/14d-Repeated dose toxicity: oral.002)

- Rationale for animal assignment (if not random): Animals were assigned to groups by the stratified randomization on the basis of body weight measured on the day before the initial dosing.

- Rationale for selecting satellite groups: Five males and 5 females (non-mating satellite females) each in the control and high dose groups were subjected to a recovery period for 14 days after the end of the dosing period.

- Post-exposure recovery period in satellite groups: 14 days

Positive control:

no

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed twice a day (before dosing and after dosing) during the dosing period and once a day in the other periods.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the following days. Measurement was performed before dosing during the dosing period. The final body weight was also measured on the day of scheduled necropsy.
‐ Test males: Days 1, 8, 15, 22, 29, 36, and 42 during the dosing period
‐ Recovery males: Days 1, 8, 15, 22, 29, 36, and 42 during the dosing period; Days 43, 50, and 56 during the recovery period
‐ Test females: Days 1, 8, and 15; Once every 7 days after the initiation of cohabitation; GDs 0, 7, 14, and 20; LDs 0, 4, 7, and 13
‐ Satellite females: At the same frequency as the males
‐ Non-copulated females: Once every 7 days after the initiation of cohabitation

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
(1) Examination time points
‐ Test males: Day 43
‐ Test females: LD 14
‐ Recovery males: Day 57
‐ Satellite females: Day 57
- Anaesthetic used for blood collection: Yes, Intraperitoneal injection of thiopental sodium
- Animals fasted: Yes
- How many animals:
‐ Test males: 5 animals per group with the smallest animal numbers
‐ Test females: 5 animals per group from the earlier parturition date with the smallest animal numbers
‐ Recovery males: All males
‐ Satellite females: All females

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: same as "HAEMATOLOGY"
- Animals fasted: Yes
- How many animals: same as "HAEMATOLOGY"

PLASMA/SERUM HORMONES/LIPIDS: Yes; Hormone concentration (total T4) analysis

- Time of blood sample collection: same as "HAEMATOLOGY"
- Animals fasted: Yes
- How many animals: Parental males only


URINALYSIS: Yes; males only.
- Time schedule for collection of urine: Day 41: urinalysis by reagent strip method (qualitative tests)
According to these results, no treatment-related changes were noted in any parameter. Therefore, urinary sediment tests and urinalysis using pooled urine samples during the dosing period and urinalysis during the recovery period were not performed.
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Functional tests: (1) Sensory reactivity to stimuli (2) Grip strength measurement
- Motor activity measurement

(1) Examination time points
The examinations were performed following the clinical observation after dosing during
the dosing period.

(2) Selection of animals:
‐ Test males: Once in the afternoon in the final week (Week 6) of the dosing period
‐ Test females: Once in the afternoon of the final week during the lactation period

According to these results, the functional tests and motor activity measurement were not
performed during the recovery period because no treatment-related change was noted
during the dosing period.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

(1) Examination time points
‐ Test males: Day 43
‐ Test females: LD 14
‐ Recovery males: Day 57
‐ Satellite females: Day 57
‐ Non-copulated females: After 14 days from the completion of the mating period

(2) Euthanasia and necropsy
All surviving animals were euthanized by exsanguination under anesthesia and all organs and tissues were immediately examined macroscopically.

Organ weights:
Selection of animals
‐ Test males: 5 animals per group with the smallest animal numbers. Testis and epididymis weights were measured in all males.
‐ Test females: 5 animals per group from the earlier parturition date with the smallest animal numbers
‐ Recovery males: All males
Satellite females: All females

After the scheduled necropsy, the organs were weighed (absolute weight) and the ratios of the organ weights to body weight (relative weight) was calculated on the basis of the body weight measured on the day of necropsy. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
(1) Selection of animals
‐ Test males: 5 animals per group with the smallest animal numbers in the control and high dose groups
‐ Test females: 5 animals per group from the earlier parturition date with the smallest animal numbers in the control and high dose groups

(2) Preparation of histopathological specimens and histopathological examination

After necropsy, the organs/tissues were fixed and preserved in 10 vol% phosphate buffered formalin solution; however, the testes and epididymides were
fixed in Bouin’s solution and the eyeballs were fixed in Davidson’s solution, and they were preserved in 10 vol% phosphate buffered formalin solution.
The organs/tissues of the test males and females and all gross lesions were embedded in paraffin, sectioned, and stained with hematoxylin and eosin, and then examined by microscopy.

No additional examination was performed because there was no test substance-related change in any organ or tissue.

Statistics:

Statistical analysis was performed with a computer system (tsPharma LabSite, Fujitsu Limited). However, a statistical analysis system EXSUS Version 8.1.0 (CAC Croit Corporation, statistical analysis software: SAS 9.4 [SAS Institute Japan Inc.]) was used for the following items: urinalysis (results from reagent strip method) and sex ratio. In all cases except for Bartlett’s test, levels of p<0.01 (1%) and p<0.05 (5%) were considered to be significant and two-tailed test was used. The data of offspring were handled on a litter-basis. The body weights after initiation of mating in non-copulated females were excluded from the evaluation.

Multiple comparison test: For the following numeral data, mean values and standard deviations were calculated in each group. Bartlett’s test was performed to compare variances among groups (significance level: 5%). When variance of data was homogeneous, Dunnett’s multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel’s multiple comparison test was performed to compare with the control group. For results from reagent strip method in urinalysis during the dosing period, Steel’s multiple comparison test was performed after the grades were converted into numeric values. Number of rearing, grip strength, motor activity, body weights, food consumption, urinalysis, hematology, blood chemistry, absolute and relative organ weights.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormality was noted in any animal throughout the dosing and recovery periods

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in males or females between the control
and test substance groups throughout the dosing and recovery periods (Table 5)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in food consumption was noted in females at 330 mg/kg
on LD 0. However, this was not judged to be treatment-related because there was no
dose-dependency.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, a statistically significant decrease in lymphocyte count
was noted in males at 330 mg/kg bw/day. However, this was not judged to be treatment-related
because there was no dose-dependency.
At the end of the recovery period, statistically significant increases in eosinophil ratio and
monocyte ratio were noted in males at 1000 mg/kg bw/day. However, these were not judged to
be treatment-related because each individual value was within or slightly out of the range
of the control group and similar changes were not noted at the end of the dosing period (Tables 8, 9)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, a statistically significant decrease in glucose was noted in
males at 1000 mg/kg. However, this was not judged to be treatment-related because each
individual value was within or slightly out of the range of the control group and no related
change was noted in any other examination. A statistically significant increase in γGT
was noted in females at 330 mg/kg. However, this was not judged to be treatment-related
because there was no dose-dependency.
At the end of the recovery period, a statistically significant decrease in albumin was noted
in males at 1000 mg/kg. Statistically significant increases in K and total cholesterol were
note in males and females at 1000 mg/kg, respectively. However, these were not judged
to be treatment-related because each individual value was within or slightly out of the range
of the control group and similar changes were not noted at the end of the dosing period (Table 10, 11)

Endocrine findings:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in parental males at the end of the dosing period between the control and test substance group for plasma total T4 concentration. (Summary attached)

Urinalysis findings:

no effects observed

Description (incidence and severity):

In qualitative tests, no statistically significant difference was noted in males between the
control and test substance groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment related changes in sensory reactivity to stimuli, grip strength and motor activity

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, statistically significant differences were noted in females
at 1000 mg/kg as follows: a low value of absolute brain weight, a high value of absolute
ovary weight, and a high value of relative thyroid weight. However, these were not
judged to be treatment-related because each individual value was within or slightly out of
the range of the control group and no related change was noted in any other examination.
A statistically significant increase in absolute seminal vesicle weight was noted in males
at 330 mg/kg. However, this was not judged to be treatment-related because there was
no dose-dependency.
At the end of the recovery period, statistically significant decreases in relative heart and
kidney weights were noted in males at 1000 mg/kg. However, these were not judged to
be treatment-related because each individual value was within or slightly out of the range
of the control group and no similar changes were noted at the end of the dosing period. (Table 14-17)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the dosing period, dark reddish patches on mucosa of the glandular stomach
were noted in 1 female in the control group, 4 females in the 110 mg/kg group, 2 females
in the 330 mg/kg group, and in 1 male in the 1000 mg/kg group. However, these were
not judged to be treatment-related because there was no clear dose-dependency in the
incidence. Situs inversus viscerum in the abdominal and thoracic cavity was noted in 1
female in the 1000 mg/kg group. However, this was not judged to be treatment-related
because this finding is a congenital anomaly.
At the end of the recovery period, dark reddish patches on mucosa of the glandular stomach
were noted in 1 female each in the control and 1000 mg/kg groups. Whitish patches on
mucous of the forestomach were noted in 1 female in the 1000 mg/kg group. However,
these were not judged to be treatment-related because there was no clear dose-dependency
in the incidence. (Tables 12, 13)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Various histopathological changes were noted in both sexes of the control and test
substance groups. However, these were not judged to be treatment-related because they
are noted occasionally in normal rats and the incidence of the findings noted in the test
substance groups was not clearly different from that in the control group.
Dark reddish patches on mucosa of the glandular stomach noted at necropsy were
hemorrhage of mucosa or erosion of the glandular stomach in histopathology. Whitish
patches on mucosa of the forestomach noted at necropsy were cysts on squamous of the
forestomach in histopathology (Table 18, 19)

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No treatment-related change was noted in males or females in any examination

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the findings of this combined repeated dose and reproduction/developmental toxicity screening test in Crl:CD(SD) rats, the NOAEL (male/female) of the test item for repeated dose toxicity is considered to be 1000 mg/kg bw/day, as no treatment-related adverse changes were noted.

Executive summary:

In a combined repeated dose and reproduction/developmental toxicity screening test (P200484), the test item was administered to 4 groups of Crl:CD (SD) rats by gavage in 0.5% w/v methylcellulose solution at dose levels of 0 (7 males, 12 females), 110 (12 males/females), 330 (12 males/females), and 1000 mg/kg bw/day (7 males, 12 females), 7 days per week. Males were dosed from 14 days before mating until day before the necropsy throughout the mating period (42 days in total). Females were dosed from 14 days before mating until Day 13 of lactation (day of delivery was designated as Day 0 of lactation) throughout the mating and gestation periods and delivery. Non-copulated females were kept until day before the necropsy. Five males and 5 females (non-mating satellite females) each in the control and high dose groups were subjected to a recovery period for 14 days after the end of the dosing period.

 

Concentration analysis and homogeneity of formulation samples was determined at three concentrations, 11 mg/mL, 32mg/mL and 100 mg/mL at first dosing formulation preparation. All samples were homogeneous. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10% (102.5-104.8%).

 

No treatment-related change was noted in any of the examinations: clinical observation, functional observational battery, body weight measurement, food consumption measurement, urinalysis in males, haematology, blood chemistry, necropsy, organ weight measurement, histopathological examination, or T4 hormone concentration measurement.

 

Based on the findings of this study the NOAEL (male/female) of the test item for repeated dose toxicity is 1000 mg/kg bw/day.