Dimethyl (p-methoxybenzylidene)malonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar rats - HsdHan: WIST rats; Conventionally bred (In-house random bred)

Sex:

male/female

Details on test animals or test system and environmental conditions:

No. of groups: 6
Main groups:
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)
Recovery Groups:
Vehicle control recovery (G1R)
High dose recovery (G4R)
No. of animals for acclimatization: 60 males and 60 females
No. of rats/group: Main groups: 10 males + 10 females per group; Recovery groups: 5 males + 5 f
emales per group; Total = 100 (50 males + 50 females)
Age at treatment: 16-17 weeks
Body weight range at the start of treatment: Males: 370.61 to 478.55 g; Females: 235.23 to 310.02 g

Route of administration:

oral: gavage

Vehicle:

other: 1 % (w/v) Na CMC (medium viscosity) in Milli-Q water

Details on oral exposure:

Males: The dose formulation was administered to the rats of the specific groups once daily at appr
oximately the same time each day (varying by +/- 3 hours) for 2 weeks prior to mating and the treat
ment was continued during the mating period and approximately two weeks post mating.
Females: The dose formulations were administered to the specific group of rats once daily at ap
proximately the same time each day (varying by +/- 3 hours) throughout the treatment period. Treatm
ent was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to
lactation day 13, after which, pups were sacrificed on lactation day 13 and parental females (dams)
were sacrificed on lactation day 14 after overnight fasting (water allowed).
The dose formulations were administered to the high dose recovery group of rats once daily at
approximately the same time each day (varying by +/- 3 hours).
The volume of dose administered was 10 mL/kg Bwt throughout the study. The dose volume was
adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at
10 mL/kg Bwt.
The vehicle and the test item was not administered for vehicle control recovery and high dose
recovery groups, respectively for 14 days following the treatment period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring
using a magnetic stirrer.

Details on mating procedure are provided in chapter 7.8.1

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DOSE FORMULATION PREPARATION:
The dose formulations were prepared daily prior to start of the treatment and prepared on need basis
and used within the stability period.
Following procedure was followed when 150 mL of dose formulation prepared:
The quantities of 1500 (G2), 4500 (G3) and 15000 (G4) mg of test item was weighed in a glass
beaker and transferred to the mortar. The test item was ground to a fine powder in the mortar using
pestle. Small volume of vehicle (1 % (w/v) NaCMC in Milli-Q water) was added in a stepwise manner
to mortar with continuous trituration till a uniform suspension was obtained. The micture was tr
ansferred into the measuring cylinder. Further, a snall volume of vehicle was added to the mortar and
rinse with vehicle to ensure the test item was completely transferred. The final volume was made up
with vehicle and to attain a desired concentration.
DOSE FORMULATION ANALYSIS:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples
were sampled in duplicate sets on Day 1 and once during Week 4 of the treatment and were sent to
Analytical R&D of Advinus Therapuetics Limited. For each set, duplicate samples were drawn from
top, middle and bottom layers of each preparation and in case of control, duplicate samples from
only middle layer was drawn.
The analysis was done as per the method validated under Advinus Study No. G9993. One set of s
amples were analyzed for concentration analysis and other set (second set) of samples were stored
at ambient conditions for reanalysis purpose as a backup and the second set of samples were
discarded, as the analysis results of first set of samples were within the limits.

Duration of treatment / exposure:

see 'Details on exposure'

Frequency of treatment:

see 'Details on exposure'

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control group (G1)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Low dose group (G2)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group (G3)

Dose / conc.:

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

Remarks:

High dose group (G4)

No. of animals per sex per dose:

10 males + 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

SELECTION OF DOSE LEVELS AND DOSE JUSTIFICATION
Three dose levels of 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day were selected for groups G2, G3 and G4 respectively as recommended by the sponsor.
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and recovery were handled in a manner similar to the treatment groups
except for test item administration

Observations and examinations performed and frequency:

Clinical Signs, Morbidity and Mortality
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs, the observation for morbidity and mortality was carried
out once in the morning during holidays. All rats were observed for clinical signs once daily during the treatment and recovery periods.

Detailed Clinical Examination
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment and recovery periods. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous
membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling or bizarre behaviour like self-mutilation, walking backwards. On the days of detailed clinical examination, general clinical signs were not performed except on
treatment Day 1.

Functional Observation Battery Tests (FOB)
The following neurological examination was conducted for randomly selected 5 males and 5 females in each group at termination i.e. towards the end of the dosing period for males (shortly prior to their scheduled kill) and during lactation period for females (shortly before the scheduled kill). For recovery groups, neurological examination was carried out towards the end of recovery period.

Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations and convulsions.

Observations during Removal of Animal from Home Cage and Handling
The objective of this phase of neurological examination was to observe the subject's response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being
held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.
Open Field Observation
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and
observed for at least 2 minutes. Absorbent paper was replaced for each rat. During this observation
period, rat was evaluated as it moves about freely/unperturbed and the following observations were
made and observations were recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- urination
- defecation
- rearing
- abnormal vocalizations

Functional Tests
Functional testing included motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.

Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in secobds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, ambulatory counts
were monitored. The Opto-Varimex 4 motor activity measurement system provides the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.

Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat is in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/
ranks.
- approach response
- touch response
- click response
- tail-pitch response
- pupil response
- aerial righting reflex
Landing Hindlimbs Footsplay
The landing hind limbs foot splay was performed by dropping the rat on to a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The heel portion of each hind foot of each rat was marked with non-permanent ink or the hind feed of the rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which has the details such as
Study No., Animal No., Group and sex. A clean recording paper sheet was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values were presented in the report.

Grip performance
Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). At least three trials were conducted for each rat i.e., three trials for forelimb and hind limbs. Average of three trials for both forelimb and hindlimbs were calculated and presented in the report.

Physiological Observations
Body temperature (rectal temperature) was measured in Celcius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.

Body weights
i) Individual body weights of males were recorded initially and at weekly intervals thereafter. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13.

Food consumption
Cagewise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period. Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of lactation period.

Parturition
The duration of gestation was calculated from Day '0' of pregnancy to the day of parturition (Gestation Length). Females were observed for signs of difficult or prolonged parturition.

Hormone Analysis
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 (Thyroxin) and thyroid stimulating hormone (TSH):
- Two pups per litter on Day 4 after birth
- All dams at termination on Day 14
- Two pups per litter on Day 13 and
- All adult males, at termination.
The blood from pups was pooled by litter for thyroid hormone analysis. Pups were lightly anesthetized with isoflurane and incised at jugular vein in the neck region. The collected samples were pooled together for each litter. Blood samples were collected in plain labeled tubes and kept on bench top for 15 -20 min before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4 °C. the serum samples were placed in labeled
plastic tubes and stored at ~-70 °C until they were analyzed.

Clinical Pathology (i.e. Blood Collection, Haematology, Coagulation Parameters, Clinical Chemistry,
Urinalysis, Thyroid Profile Hormones) as well as Anatomic Pathology (i.e. Necropsy, Tissue Collection,
Histopathology) were also assessed within this study.

Sacrifice and pathology:

Postmortem examinations (parental animals)
All adult animals were examined macroscopically for any structural abnormalities and pathological changes. The adult animals sacrificed at term were fasted overnight (water allowed), exsanguinated under isoflurane anaesthesia and weighed. The rats were subjected for detailed necropsy by a veterinary pathologist and findings were recorded. The number of implantation sites was recorded for all the dams.

Statistics:

The statistical analysis of the experimental data was carried out using the validated package in excel and also using licensed copies of SYSTAT Statistical package ver. 12.0. All quantitative variables like body weight, food intake, terminal body weight, clinical pathology (haematology clinical chemistry a nd coagulation) parameters, thyroid hormone levels, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Nonoptimal (non-normal or heteroscedastic) data were transformed, before using ANOVA. Means between treatment groups and vehicle control group were compared using Dunnett's test. Pre-implantation loss (%), post-implantation loss (%), no. of corpora lutea, implantations, pre-coital interval, sex ratio (%) and gestation length (days) were analysed after suitable transformation of the data. One-way ANOVA was carried out for the transformed data. Dunnett's pair-wise comparison of the treated means with the control mean was done for the significant group differences. Z test was performed for testing the differences in proportions for mating and fertility indices.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed at any of the doses tested. However, clinical sign of sparse hair loss at right and left flank was observed in a female (Rt1396) of control group and one male rat (Rt1473) of high dose recovery group was considered spontaneous finding and not related to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed at any of the doses tested.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males, body weights: No treatment related variations in the mean body weights at all the dose groups during the treatment period. The mean body weight was significantly lower in high dose recovery animals.
Females, body weights: No treatment related variations in the mean body weights at all the dose groups either during the treatment period or recovery periods.
Maternal Body Weights and Food Intake during the Gestation Period: No significant changes in maternal body weight and food consumption were observed at any of the doses tested when compared to the vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: The Food consumption was unaffected by the treatment at 100 mg/kg bw/day dose groups. At 300 mg/kg bw/day, the food consumption was significantly lower on week 6 and was considered in cidential because of isolated occurrence and there was no associated change in the body weight. At 1000 mg/kg bw/day, the food consumption was significantly lower and considered treatment related finding. In the 1000 mg/kg bw/day dose recovery group, the food consumption was also significantly lower on weeks 2 and 3 and hence were considered to be transient and not of biological significance.
Females: The food consumption was unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. In the high dose recovery group, the food consumption was significantly lower on weeks 1 and 3 and was considered incidential because of isolated occurrence and there was no associated change in the body weight.

Haematological findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the hematology parameters of both male and female rats.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the clinical chemistry parameters of both male and female rats.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the urinalysis parameters of both male and female rats.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Males and females: Home cage, Handling, Open field and Sensory observations: No treatment-related abnormalities were observed in all the tested dose groups either during treatment period or recovery periods.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At 1000 mg/kg bw/day, the test item-related changes included the following: Decreased terminal fasting body weight in males, decreased prostate and seminal vesicle with coagulating gland weights without any associated microscopic changes, considered secondary to reduction in body weight; increased kidney weight in males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, the test item-related changes included the following: grossly white foci in nonglandular mucosa of stomach in male rats. There were no test item-related changes in male and female reproductive organs.

Neuropathological findings:

no effects observed

Description (incidence and severity):

MALES:
-Neuromuscular observations: No treatment-related variations in the hind limb footsplay and forelimbs and hind limbs grip strength either during the treatment period or recovery periods.
-Motor activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:
Lower: Stereotypic time at interval 1, interval 3 and total at the mid dose; Ambulatory time at interval 1 at the high dose recovery. The above observed statistical variations in the motor activity measurement were considered to be incidental as the observed changes did not follow the dose proportion and there were no changes in the home cage of open field observations.
FEMALES:
- Neuromuscular observations: No treatment-related variations in the hind limb footsplay and forelimbs and hind limbs grip strength either during the treatment period or recovery periods. However, higher hindlimbs footsplay value was observed at mid dose group and was considered incidential be cause of the lack of any dose relationship.
- Motor activity: No treatment-related changes were observed in the motor activity at all the tested
doses.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related histopathological changes were observed in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa; and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration) in both sexes at 1000 mg/kg bw/day, This change showed complete recovery at the end of recovery period. There were no test item-related changes in male and female reproductive organs.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.
No treatment-related effects were observed with respect to mean pre-coital time, gestation length and fertility indices when compared to control means.
A significantly lower female fertility index at 100 and 1000 mg/kg bw/day doses was considered toxicologically insignificant due to lack of dose relationship.
No treatment-related effects were observed with respect to corpora lutea, implantations, and percentage of pre and post implantation losses when compared to control means.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Under the conditions of this OECD 422 study, the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

Executive summary:

In this GLP-compliant study according to OECD guideline 422 the test item was administered daily (gavage) to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females).

Based on the changes observed in both sexes at 1000 mg/kg bw/day, i.e. reduced body weights and food consumption, prostate and seminal vesicle with coagulating gland weights, increased kidney weight and grossly white foci in nonglandular mucosa of stomach in male rats, microscopic changes in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration)  the no observed adverse effect level (NOAEL) for systemic toxicity is considered to be 300 mg/kg bw/day.

The test item did not induce any adverse effects on fertility and reproductive perfomance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

Limit test:

no

Justification for study design:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects on male and female reproduction performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl (p-methoxybenzylidene)malonate

EC Number:

231-185-8

EC Name:

Dimethyl (p-methoxybenzylidene)malonate

Cas Number:

7443-25-6

Molecular formula:

C13H14O5

IUPAC Name:

1,3-dimethyl 2-[(4-methoxyphenyl)methylidene]propanedioate

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats - HsdHan: WIST rats; Conventionally bred (In-house random bred)

Sex:

male/female

Details on test animals or test system and environmental conditions:

No. of groups: 6
Main groups:
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)

Recovery Groups:
Vehicle control recovery (G1R)
High dose recovery (G4R)

No. of animals for acclimatization: 60 males and 60 females

No. of rats/group: Main groups: 10 males + 10 females per group; Recovery groups: 5 males + 5 females per group; Total = 100 (50 males + 50 females)

Age at treatment: 16-17 weeks

Body weight range at the start of treatment: Males: 370.61 to 478.55 g; Females: 235.23 to 310.02 g

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % (w/v) Na CMC (medium viscosity) in Milli-Q water

Details on exposure:

TREATMENT:
Males: The dose formulation was administered to the rats of the specific groups once daily at approximately the same time each day (varying by +/- 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating period and approximately two weeks post mating.
Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by +/- 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to lactation day 13, after which, pups were sacrificed on lactation day 13 and parental females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed).
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by +/- 3 hours).
The volume of dose administered was 10 mL/kg Bwt throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 10 mL/kg Bwt.
The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring using a magnetic stirrer.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there is evidence of sperms in the vaginal smear and/or vaginar plug or for a maximum period of 2 weeks. Subsequently, pregnant females were housed individually until lactation day 14. The females not mated within 14 days of pairing with the first male was placed with a second proven male of the same group and the presence of sperm in the vaginal smear and/or vaginal plug is not confirmed within 7 days, that female was sacrificed 24-26 days after last day of mating period. All the mated females were maintained till they litter or for a maximum period of at least 25 days from the last day of cohabitation. Not littered females were sacrificed after 25 days from the day they are found of sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day 0 (GD 0). The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DOSE FORMULATION PREPARATION:
The dose formulations were prepared daily prior to start of the treatment and prepared on need basis and used within the stability period.
Following procedure was followed when 150 mL of dose formulation prepared:
The quantities of 1500 (G2), 4500 (G3) and 15000 (G4) mg of test item was weighed in a glass beaker and transferred to the mortar. The test item was ground to a fine powder in the mortar using pestle. Small volume of vehicle (1 % (w/v) NaCMC in Milli-Q water) was added in a stepwise manner to mortar with continuous trituration till a uniform suspension was obtained. The micture was transferred into the measuring cylinder. Further, a snall volume of vehicle was added to the mortar and rinse with vehicle to ensure the test item was completely transferred. The final volume was made up with vehicle and to attain a desired concentration.

DOSE FORMULATION ANALYSIS:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and once during Week 4 of the treatment and were sent to Analytical R&D of Advinus Therapuetics Limited. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control, duplicate samples from only middle layer was drawn.
The analysis was done as per the method validated under Advinus Study No. G9993. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient conditions for reanalysis purpose as a backup and the second set of samples were discarded, as the analysis results of first set of samples were within the limits.

Duration of treatment / exposure:

see 'Details on exposure'

Frequency of treatment:

see 'Details on Exposure'

Details on study schedule:

see 'Details on Exposure'

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control group (G1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low dose group (G2)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid dose group (G3)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose group (G4)

No. of animals per sex per dose:

10 males + 10 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

SELECTION OF DOSE LEVELS AND DOSE JUSTIFICATION
Three dose levels of 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg bw/day were selected for groups G2, G3 and G4 respectively as recommended by the sponsor.
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and recovery were handled in a manner similar to the treatment groups except for test item administration.

Examinations

Parental animals: Observations and examinations:

See in detail under "Any other information on materials and methods...", below

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. Vaginal smears were also monitored daily from the beginning of the treatment period until evidence of mating. The time interval (in days) from the diestrus of an oestrus cycle to the next diestrus was considered as the oestrus cycle length of an animal. The overall pattern of each female was characterized as regularly cycling (having recurring 4-5 day cycles).
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on Days 0, 4 and 13 post-partum were recorded.
c. On Day 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Blood samples was collected from two of the surplus pups, pooled, and used for determination of serum T4 levels. If there were insufficient pups in a litter to have two surplus pups, two of the pups that were typically retained were used for blood collection for serum T4 assessments. When litter size dropped below the culling target (8 pups/litter), preference was given to remove two female pups for PND 4 blood samples in order to retain more male pups for observation of nipple retention on post-natal day (PND) 13.
d. After standardization, the individual pup body weight was recorded on Day 13 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on postnatal day (PND) 0 and pup body weight was recorded.
f. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
g. Fertility index for dams, sires as well as the pup survival index till LD 4 was calculated.

Postmortem examinations (parental animals):

All adult animals and pups were examined macroscopically for any structural abnormalities and pathological changes. The adult animals sacrificed at term were fasted overnight (water allowed), exsanguinated under isoflurane anaesthesia and weighed. The rats were subjected for detailed necropsy by a veterinary pathologist and findings were recorded. All the surviving pups were necropsied on LD 13 and findings were recorded. Dead pups were examined for possible defects and/or cause of death. The number of implantation sites was recorded for all the dams.

Statistics:

The statistical analysis of the experimental data was carried out using the validated package in excel and also using licensed copies of SYSTAT Statistical package ver. 12.0. All quantitative variables like body weight, food intake, terminal body weight, clinical pathology (haematology clinical chemistry and coagulation) parameters, thyroid hormone levels, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroscedastic) data were transformed, before using ANOVA. Means between treatment groups and vehicle control group were compared using Dunnett's test.
Pre-implantation loss (%), post-implantation loss (%), no. of corpora lutea, implantations, pre-coital interval, sex ratio (%) and gestation length (days) were analysed after suitable transformation of the data. One-way ANOVA was carried out for the transformed data. Dunnett's pair-wise comparison of the treated means with the control mean was done for the significant group differences.
Z test was performed for testing the differences in proportions for mating and fertility indices.
Statistically significant differences (p <= 0.05), indicated by the aforementioned tests was designated throughout the report as stated below:
+/- : Significantly higher (+) / lower (-) than the vehicle control group
a+/a- : Significantly higher (a+) / lower (a-) than the vehicle control recovery group

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed at any of the doses tested. However, clinical sign of sparse hair loss at right and left flank was observed in a female (Rt1396) of control group and one male rat (Rt1473) of high dose recovery group was considered spontaneous finding and not related to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed at any of the doses tested.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males, body weights: No treatment related variations in the mean body weights at all the dose groups during the treatment period. The mean body weight was significantly lower in high dose recovery animals.
Females, body weights: No treatment related variations in the mean body weights at all the dose groups either during the treatment period or recovery periods.

Maternal Body Weights and Food Intake during the Gestation Period: No significant changes in maternal body weight and food consumption were observed at any of the doses tested whn compared to the vehicle control group.

Maternal Body Weights and Food Intake during the Lactation Period: No significant changes in maternal body weight and food consumption were observed at any of the doses tested when compared to the vehicle control group. However, at 1000 mg/kg bw/day, the body weight change was significantly higher during the Days 0-13 and considered incidential as there were no change in the mean body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: The Food consumption was unaffected by the treatment at 100 mg/kg bw/day dose groups. At 300 mg/kg bw/day, the food consumption was significantly lower on week 6 and was considered incidential because of isolated occurrence and there was no associated change in the body weight. At 1000 mg/kg bw/day, the food consumption was significantly lower and considered treatment related finding. In the 1000 mg/kg bw/day dose recovery group, the food consumption was also significantly lower on weeks 2 and 3 and hence were considered to be transient and not of biological significance.

Females: The food consumption was unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. In the high dose recovery group, the food consumption was significantly lower on weeks 1 and 3 and was considered incidential because of isolated occurrence and there was no associated change in the body weight.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Males & Females - Home cage, Handling, Open field and Sensory observations: No treatment-related abnormalities were observed in all the tested dose groups either during treatment period or recovery periods.

Description (incidence and severity):

MALES:
-Neuromuscular observations: No treatment-related variations in the hind limb footsplay and fore limbs and hind limbs grip strength either during the treatment period or recovery periods.
-Motor activity: The following statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group:
Lower: Stereotypic time at interval 1, interval 3 and total at the mid dose; Ambulatory time at interval 1 at the high dose recovery.
The above observed statistical variations in the motor activity measurement were considered to be incidental as the observed changes did not follow the dose proportion and there were no changes in the home cage of open field observations.

FEMALES:
- Neuromuscular observations: No treatment-related variations in the hind limb footsplay and fore limbs and hind limbs grip strength either during the treatment period or recovery periods. However, higher hindlimbs footsplay value was observed at mid dose group and was considered incidential because of the lack of any dose relationship.
- Motor activity: No treatment-related changes were observed in the motor activity at all the tested doses.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation). The calculated mean oestrous cycle length was 4.49, 4.58, 4.57 and 4.79 days in vehicle control, 100, 300 and 1000 mg/kg bw/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Fertility index:
No treatment-related effects were observed with respect to mean pre-coital time, gestation length and fertility indices when compared to control means.
the significantly lower male mating and fertility indices at all the doses tested were observed when compared to vehicle control group. These changes were considered incidential, as there changes were marginal and within the in-house historical control data range (Male mating index: 90-100 %; Male fertility index: 80 - 100 %).
The significantly lower female fertility index at 100 and 1000 mg/kg bw/day doses was considered toxicologically insignificant due to lack of dose relationship.

Uterine/Implantation Data:
No treatment-related effects were observed with respect to corpora lutea, implantations, and percentage of pre and post implantation losses when compared to control means.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

haematology

clinical biochemistry

urinalysis

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: NOAEL for reproductive performance

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Remarks on result:

other: NOAEL for systemic toxicity

Results: P1 (second parental generation)

General toxicity (P1)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival Data of Pups:
Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter size, and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.
No treatment-related changes were observed in the survival data of pups up to LD 4 at 100 and 300 mg/kg bw/day. However, lower survival data of pups observed on Day 4 at 1000 mg/kg bw/day which was considered to be due to dams Rt1456, where partial pups were dead and Rt1457, where all pups were dead and cannibalized during LDs 1-2. These isolated changes that can be observed spontaneously in rats of this strain and were considered to be incidential.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Number and Body Weight of Pups during the Lactation Period:
The mean number of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 1000 mg/kg bw/day, the mean number of total pups per litter was significantly lower on lactation Days 4 and 13.
The mean weight of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 100 mg/kg bw/day, the mean weight of total pups per litter were significantly higher on lactation Day 13 and considered to be incidential because of the lack of dose relationship.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Ano-genital Distance (AGD):
No changes attributable to test item were detected in the AGD, body weight and ratio of AGD to the cube root of body weight of either sex.
However, the body weight of male pups was significantly lower at 300 mg/kg bw/day when compared to control group and considered to be incidential because of the lack of dose relationship.
The body weights of female pups were significantly lower at 300 and 1000 mg/kg bw/day when compared to control group.

Areolae/Nipple retention in Pups:
The male pups did not exhibit areola/nipple retention on PND 13.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Survival Data of Pups:
Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter size, and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups.
No treatment-related changes were observed in the survival data of pups up to LD 4 at 100 and 300 mg/kg bw/day. The mean number of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 1000 mg/kg bw/day, the mean number of total pups per litter was significantly lower on lactation Days 4 and 13. However, lower survival data of pups observed on Day 4 at 1000 mg/kg bw/day which was considered to be due to dams Rt1456, where partial pups were dead and Rt1457, where all pups were dead and cannibalized during LDs 1-2. These isolated changes that can be observed spontaneously in rats of this strain and were considered to be incidential.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight of male pups on lactation day 0 was significantly lower at 300 mg/kg bw/day when compared to control group and considered to be incidential because of the lack of dose relationship.
The body weights of female pups on lactation day 0 were significantly lower at 300 and 1000 mg/kg bw/day when compared to control group, but not clearly dose-related. However, this effect was compensated during lactation and the body weights were no longer significantyl altered at end of lactation:
The mean weight of male, female and total pups per litter were unaffected by the treatment at 100, 300 and 1000 mg/kg bw/day dose groups. However, at 100 mg/kg bw/day, the mean weight of total pups per litter were significantly higher on lactation Day 13 and considered to be incidential because of the lack of dose relationship.

Other effects:

no effects observed

Description (incidence and severity):

Ano-genital Distance (AGD):
No changes attributable to test item were detected in the AGD, body weight (see above) and ratio of AGD to the cube root of body weight of either sex.
Areolae/Nipple retention in Pups:
The male pups did not exhibit areola/nipple retention on PND 13.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

other: Anogenital distance and nipple retention

Remarks on result:

other: NOAEL for developmental effects

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

- Daily oral (gavage) administration of the test item to male and female Wistar rats at dose levels of 100, 300 and 1000 mg/kg bw/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) did not induce any adverse effects on fertility and reproductive perfomance. The No Observed Adverse Effect Level (NOAEL) of the test item is considered to be 1000 mg/kg bw/day.

-Considering the changes observed in the body weights and food consumption, prostate and seminal vesicle with coagulating gland weights, increased kidney weight and grossly white foci in nonglandular mucosa of stomach in male rats, microscopic changes in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration) in both sexes at 1000 mg/kg bw/day, the no observed adverse effect level (NOAEL)" for systemic toxicity is considered to be 300 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study was provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was suspended in vehicle [1 % (w/v) Carboxymethyl cellulose (CMC) sodium salt in Milli-Q water] and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg bw/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. A concurrent control (G1) and a control recovery group (G1R) of rats received vehicle alone. The dose volume administered was 10 mL/kg bw/day. The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The prepared dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to lactation day 13 (for females). In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams.

The identity of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No. G9993. Based on the results, the test item was found to be stable for up to 9 days at 1 and 100 mg/mL concentrations when stored at room temperature and refrigeration condition. The dose formulations were analysed for active ingredient (a. i.) concentration on Day 1 and during Week 4 of the treatment period. The results indicated that the analysed concentrations were within +/- 15 % of variations from the nominal concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7, 11 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were exampined for the appearance nipples/aerolae on PND 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group. For recovery groups, functional observation battery was done prior to sacrifice. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. T4 and TSH hormone analysis was performed in all males at termination, all dams at termination on LD14 and available two pups per litter on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues collected from randomly selected 5 males and 5 females in the control and high dose groups were examined microscopically for histopathological changes. A gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland from available one male and one female pup from each litter was collected for histopathological examination. The reproductive organs of infertile males (Rt1404, Rt1429 and Rt1443) and females (Rt1419 and Rt1460) rats were examined microscopically.

Under the experimental conditions employed, the following results were obtained:

- There were no treatment-related clinical signs or mortality observed at any of the doses tested.

- No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.

- At 1000 mg/kg bw/day, the mean body weights and food consumption were significantly lower in males when compared to vehicle control group.

- The body weights and food consumption were unaffected by the treatment during gestation and lactation period at all the doses tested.

- Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

- There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. The survival index was not altered by the treatment at any of the doses tested.

- No treatment-related changes in the ano-genital distance and ano-genital index were observed at any of the doses tested when compared to the control group.

- The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

- The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females.

- At 1000 mg/kg bw/day, the terminal fasting body weight was decreased when compared to the control group.

- At 1000 mg/kg bw/day, test item-related changes includes decrease in weights of prostate and seminal vesicle with coagulating gland of males, inreased weights of ovaries with oviducts and uterus with cervix in female rats. Increased absolute and relative kidney weight in male rats were observed.

- There were no significant changes in thyroid stimulating hormone (TSH) and thyroxin hormone levels in adult animals and pups. there were no test item-related changes in thyroid weights of pups. There were no test item-related microscopic changes in thyroid gland of pups.

- Test item-related histopathological changes were observed in stomach (hyperkeratosis and epithelial hyperplasia in nonglandular mucosa; and mucosal necrosis) and kidneys (dilated tubules and tubular degeneration/regeneration) in both sexes at 1000 mg/kg bw/day, This change showed complete recovery at the end of recovery period. There were no test item-related changes in male and female reproductive organs.

- Reduction observed in absolute and relative weights of prostate and seminal vesicle with coagulating gland of males at 1000 mg/kg bw/day without any associated microscopic changes was attributed to reduced body weight observed in these rats and considered as a test item-related secondary change.