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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to Reproduction (Screening):

Subacute NOAEL (reproduction, parental): 1000 mg/kg bw/day (OECD 422/GLP)

Subacute NOAEL (development, offspring): 1000 mg/kg bw/day (OECD 422/GLP)

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, 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.

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

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% Conversion factor: 1.08 (100/92.4)


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): The test substance was administered orally using a disposable syringe attached to a gastric tube.


Purity correction was applied.

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)

Route of administration:

oral: gavage

Vehicle:

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

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: The test males and females of each dose level were cohabited at one-on-one basis day-andnight on Day 15 up to 14 days.
- Proof of pregnancy: The vaginal smears were collected in the morning from the day after the initiation of mating, and copulation was confirmed by the presence of the vaginal plug or sperm in the smear sample. The day of successful mating was regarded as Day 0 of gestation (GD 0).

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

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

330 mg/kg bw/day (nominal)

Dose / conc.:

110 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

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

Parental animals: Observations and examinations:

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.

Observation during lactation period
(1) Observation time points: Once daily from LD 0 to LD 13
(2) Observation method: The delivered animals (dams) were allowed to nurse offspring until day 13 postpartum (LD 13), and postpartum behavior such as lactation, nesting and presence or absence of cannibalism was observed every day.

Examination after lactation
(1) Examination time points
‐ Test females: At necropsy on LD 14
(2) Confirmation of the implantation sites
‐ Test females: The ovaries and uterus were excised to examine for the numbers of implantation sites.

Oestrous cyclicity (parental animals):

(1) Collection of vaginal smears
Vaginal smears were collected with swabs from all test females in the morning (same time
every day) from the initial dosing day to the day of successful copulation or end of the
mating period to confirm the estrous phase. The obtained smears were collected on a
plate for each animal, and stained with Giemsa.
(2) Classification of estrous phase
‐ Diestrus (D)
‐ Proestrus (P)
‐ Estrus (E)
‐ Metestrus (M)
(3) Parameters
‐ Mean estrous cycle:
Number of days from the estrus to the next estrus
‐ Number of the estrus during the test period

Sperm parameters (parental animals):

Parameters examined in all P male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
On PND4, the litter size was standardized to 8 (4/sex/litter) by random removal of offspring. Even
when the number of either males or females per litter was less than 4, the litter size was
adjusted to 8 regardless of the sex ratio. A litter with less than 8 offspring was maintained
as is.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups,, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
All stillborn and dead offspring were observed for external anomalies.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

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.

Postmortem examinations (offspring):

SACRIFICE
PND 13

GROSS NECROPSY
- The thoracoabdominal organs/tissues were examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid glands of 1 male and 1 female per litter (animals subjected to blood sampling for hormone concentration measurement) were collected and fixed and preserved in 10 vol % phosphate buffered formalin.

Hormone concentration (total T4) analysis
(1) Sampling time points: PNDs 4 and 13
(2) Selection of animals
‐ PND 4: Blood was collected from 2 culled offspring (1 male and 1 female or 2 males) in each litter.
‐ PND 13: Blood was sampled from 1 animal of each sex in each litter.

When a blood sample could not obtained from either sex, it was obtained from 2 animals of the same sex in the litter. As for dams with only one offspring, the blood sampling was not performed.

Statistics:

Statistical analysis:
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. Analysis for concentration of total T4 was performed according to work plan. The data of offspring were handled on a litter-basis.

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, estrous cycle, number of estrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weights of offspring, AGD, and AGI.

Chi-square test:
Copulation index, fertility index, gestation index, and sex ratio

Wilcoxon’s rank sum test:
Delivery index, stillborn index, external anomaly index, external anomaly typing index, birth index, viability index on Days 4 and 13, and nipple development anomaly index

Reproductive indices:

Gestation index (%): (Number of pregnant animals delivered live offspring / Number of pregnant animals) × 100;

Delivery index (%): (Number of delivered offspring / Number of implantations) × 100; Copulation index; Fertility index.

Offspring viability indices:

Birth index (%): (Number of live offspring at birth / Number of implantations) × 100;

Stillborn index (%): (Number of stillborns / Number of delivered offspring) × 100;

Viability index on Day 4 (%): (Number of live offspring on PND 4 / Number of live offspring at birth) × 100;

Viability index on Day 13 (%): (Number of live offspring on PND 13 / Number of live offspring after culling) × 100;

Sex ratio: (Number of male live offspring / Number of male and female live offspring);

External anomaly index (%): (Number of offspring with external anomaly / Number of observed offspring) × 100;

External anomaly typing index (%): (Number of offspring with external anomaly by each type / Number of observed offspring) × 100.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormality was noted in any animal throughout the dosing and recovery periods (Table 1)

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. 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. 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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

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

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in the number of estrus or mean estrous
cycle between the control and test substance groups. The phase of the estrous cycle at necropsy was diestrus in all dams. (Table 20)

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effect on testes or epididymides weights.

Reproductive performance:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in the copulation index, duration of mating,
or fertility index between the control and test substance groups.
No abnormality was noted in the delivery or nursing. No statistically significant
difference was noted in the gestation index, duration of gestation, number of implantations,
or delivery index between the control and test substance groups. (Table 21, 22)

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 any of the parental reproductive parameters

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No statistically significant difference was noted in the number of litter, live newborns, or
stillborns, birth index, stillborn index, sex ratio, or viability index on PND 4 or 13 between
the control and test substance groups. (Table 22)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted between the control and test substance
groups. (Table 23)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in offspring on PND 13 between the control and test substance group in total plasma T4 (summary attached).

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in the AGD or AGI between the control
and test substance groups (Table 25)

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No statistically significant difference was noted in the anomaly index between the control
and test substance groups (Table 26)

Organ weight findings including organ / body weight ratios:

not specified

Description (incidence and severity):

The thyroid glands of 1 male and 1 female per litter were collected and fixed and preserved in
10% phosphate buffered formalin but no data on weights were reported.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic change was noted in any offspring (Table 27)

Other effects:

no effects observed

Description (incidence and severity):

No abnormality was noted in any offspring on Day 0 or 13 upon external examination (Table 24)

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

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 any of the inspections for offspring.

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Conclusions:

Based on the findings of this combined repeated dose and reproduction/developmental toxicity screening test in Crl:CD(SD) rats, the NOAEL (parental, offspring) of the test item for reproductive/developmental 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 in parental animals: clinical observation, functional observational battery, body weight measurement, food consumption measurement, urinalysis in males, haematology, blood chemistry, necropsy, organ weight measurement, histopathological examination, or male parental T4 hormone concentration measurement.

 

In the parental animals, no treatment-related change was noted in any of the examinations: estrous cycle, copulation index, days until copulation, fertility index, gestation index, gestation length, numbers of implantations, delivery index, or parturition/maternal behaviour. In the offspring, no treatment-related change was noted in any of the examinations: sex ratio, birth index, stillborn index, viability index, external examination, body weight, anogenital distance, nipple development, necropsy, or plasma total T4 concentration. 

 

Based on the findings of this study the NOAEL (parental, offspring) of the test item for reproductive/developmental toxicity is 1000 mg/kg bw/day.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was the only study available and was assigned a Klimisch score of 2. The overall quality of the database is high.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to Reproduction (Screening):

There is one dose range finding (DRF) study in rats available and one Combined Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in rats available.

 

 Dose range finding (DRF) study

In a dose range finding study, for a combined repeated dose toxicity study with reproduction/developmental toxicity screening test in rats (OECD 422/GLP), the test item was administered to Crl: CD (SD) rats (5/sex/dose) by gavage in 0.5% (w/v) methylcellulose aqueous solution at dose levels of 0, 110, 330 and 1000 mg/kg bw/day for 14 days.  There was no mortality and no clinical signs were noted. There were no test item-related effects observed on body weight, food consumption or haematological parameters. No test item-related macroscopic findings were observed up to a dose level of 1000 mg/kg bw/day. Statistically significant changes were observed in total bilirubin in males and ALAT in females of the 1000 mg/kg group, but these were all slight changes compared with the control group and no other related changes were observed. Therefore, it was concluded that these changes were not related to the administration of the test substance. Statistically significant changes were observed in the absolute glans penis weight in males and spleen weight in females of the 330 mg/kg bw/day group, but these were not dose-related and were not considered to be related to the test substance. In conclusion, under the conditions of this study, after 14 days of oral gavage treatment at up to 1000 mg/kg bw/day, there were no clear adverse effects of test item treatment. Therefore, the doses selected for the main OECD 422 test were 110, 330 and 1000 mg/kg bw/day.

 

Combined Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

There is one combined repeated dose and reproduction/developmental toxicity screening test in rats available.

 

In a combined repeated dose and reproduction/developmental toxicity screening test (OECD 422/GLP), 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 in parental animals: clinical observation, functional observational battery, body weight measurement, food consumption measurement, urinalysis in males, haematology, blood chemistry, necropsy, organ weight measurement, histopathological examination, or male parental T4 hormone concentration measurement. In the parental animals, no treatment-related change was noted in any of the examinations: estrous cycle, copulation index, days until copulation, fertility index, gestation index, gestation length, numbers of implantations, delivery index, or parturition/maternal behaviour. In the offspring, no treatment-related change was noted in any of the examinations: sex ratio, birth index, stillborn index, viability index, external examination, body weight, anogenital distance, nipple development, necropsy, or plasma total T4 concentration. Based on the findings of this study the NOAEL (parental, offspring) of the test item for reproductive/developmental toxicity is 1000 mg/kg bw/day.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the information available in the dossier, the substance does not need to be classified for reproductive toxicity or for effects on or via lactation when the criteria outlined in Annex I of 1227/2008/EC are applied.