Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-11-16 to 2013-06-13
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): UNDECYLENOYL PHENYLALANINE
- Substance type: Lipoamino acid
- Physical state: White powder
- Analytical purity: Consider as 100% (dry extract)
- Purity test date: 23/03/2012
- Lot/batch No.: 1206800019
- re-test date: 08/03/2015
- Storage condition of test material: Ambient (+ 15 to +25 °C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxicology, Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, India
- Age at study initiation: 12 weeks
- Weight at study initiation: Males: 278.90 to 374.22g; Females: 183.90 to 246.47g
- Fasting period before study: No
- Housing:
Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: approximately L 425 x B 266 x H 175 mm), with stainless steel top grill having facilities for pelletted food and drinking water in polycarbonate bottles with stainless steel sipper tubes except for last animal in the recovery group wherein one animal was housed.
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum):
Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories B.V. Maasheseweg 87c PO Box 553, 5800, AN Venray, The Netherlands was provided ad libitum to the animals.
- Water (e.g. ad libitum):
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20 to 23°C
- Humidity (%): 54 to 67 %
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: November 17th, 2012 To: January 18th, 2013

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% Carboxymethyl cellulose with 0.1% Tween 80 in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Following procedure was followed when 100 mL of dose formulation prepared:
The quantities of 0.5, 1.5 and 6.0 g of test item was weighed on a separate aluminum foil and carefully transferred into separate mortar. Small aliquot of vehicle [0.5% (w/v) sodium salt of carboxymethyl cellulose (medium viscosity) with 0.1 % (v/v) Tween 80 in Milli-Q water] was added to each
mortar and triturated well to obtain the pasty mass of the suspension. Required aliquot of vehicle was added to the mass and triturated to obtain the suspension and then transferred to separate measuring cylinders. The mortar and pestle was rinsed with vehicle and it was transferred to the respective measuring cylinders. The final volume was made up with the vehicle to get the final concentration of 5, 15 and 60 mg/mL for the G2, G3 and G4/G4R groups, respectively.
The suspensions were constantly stirred using a magnetic stirrer during test item administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): As the Ti is not soluble/stable in water, 0.5% (w/v) Sodium Carboxymethyl Cellulose (medium viscosity) and 0.1% (v/v) Tween 80 in Milli-Q was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study (study no. G8471). Indeed, this vehicle was commonly used for iterative studies, moreover the test item was well solubilized in this matrix.
- Concentration in vehicle: 0.5 % (CMC) and 0.1% (Tween 80)
- Amount of vehicle (if gavage): 10 mL/Kg body weight
- Lot/batch no. (if required): CMC (Sigma) batch:SLBB5612V, Tween 80 (Sigma) bacth:BCBB5990
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: Nil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, the prepared dose suspension was sampled from each dose levels (one sample
was drawn from each of the top, middle and bottom layers) on Day 1 and during 2nd month of the treatment period and sent to Analytical R&D of
Advinus Therapeutics Limited, for analysis. Similar sample only from middle layer was drawn from vehicle control sample. The sample analyses was
done as per the analytical method validated under Advinus Study No.: G8470
Duration of treatment / exposure:
Males: The dose formulation was administered to the rats of the specific groups once daily at approximately the same time each day (varying by ± 2 hours) for 2 weeks prior to mating. Treatment was continued during mating and up to and including the day before sacrifice which was done after the completion of the mating process.
Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 2 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 4, after which, pups were sacrificed on lactation day 4 and females (dams) were sacrificed on lactation day 5 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 ± 2 hours).
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups.
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
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.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 14 - 15 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
600 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
Main groups : 10 males+10 females per group
Recovery groups : 5 males + 5 females per group
Total = 100 (50 males + 50 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels of 50 (G2), 150 (G3) and 600 (G4) mg/kg/day were selected for this study based on the results of 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No.G8471) and in consultation with the Sponsor.

In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and recovery animals were handled in a manner similar to the treatment groups except for test item administration.

- Rationale for animal assignment (if not random): Random
- Other: Nil
Positive control:
No positiv control

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.2]

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of males were recorded initially and at weekly (±1 day) intervals thereafter. Individual body weights of females were recorded initially and at weekly (±1 day) intervals thereafter till cohabitation with males. For recovery groups the individual body weight was recoded initially and weekly (±1 day) intervals thereafter.
All dams were weighed on Gestation Days (GD) 0, 7, 14 and 20 and on lactation days 0 and 4 and weights were recorded.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

Oestrous cyclicity (parental animals):
Yes, during cohabitation.
Sperm parameters (parental animals):
No
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

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, physical or behavioural abnormalities, other:]

GROSS EXAMINATION OF DEAD PUPS: Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after completion of mating and at least 4 weeks treatment
- Maternal animals: All surviving animals on day 5 of lactation

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the Study Plan were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The offspring were sacrificed on day 4 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
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, haematology, clinical chemistry, 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 ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant.

Pre-implantation loss (%), post implantation loss (%), no. of corpora lutea and implantations, pre-coital interval and gestation length (days) was analysed after suitable transformation (√ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.

Z test was performed for testing the differences in proportions for mating and fertility indices.

All analyses and comparisons were evaluated at the 5% (P≤0.05) level. Statistically significant differences (P≤0.05), indicated by the aforementioned
tests were designated by the superscripts 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
Reproductive indices:
a. Male mating index (%)
b. Male fertility index (%)
c. Female mating index (%)
d. Fecundity index (%)
e. Female fertility index (%)
f. Mean number of corpora lutea (CL)/group
g. Mean number of implantations/group
h. Implantation index
i. Percentage of pre-implantation loss per group
j. Post implantation loss (%)
k. Gestation index
Offspring viability indices:
a. Mean litter size per group
b. Live birth index (%)
c. Day 4 survival index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
the treatment resulted in decrease in the mean body weights in males when treated at 600 mg/kg Bwt/day when compared to vehicle control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
the treatment resulted in decrease in the mean body weights in males when treated at 600 mg/kg Bwt/day when compared to vehicle control.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
epithelial hyperplasia in stomach; non glandular stomach ulceration; lymphoid depletion in thymus
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

GROSS PATHOLOGY (PARENTAL ANIMALS):
The incidences of non-glandular stomach thickening at 600 mg/kgBwt/day (1/10 in males, 6/10 in females) were considered as test item related.
However, these results suggest that in females the incidences were higher than males. Moreover, at 150 mg/kgBwt/day dose group in females,
non-glandular stomach lesions were observed (2/10) and considered as test item related.
The small sized thymus observed in ≥150 mg/kgBwt/day dose group females was considered as test item related.
The microscopic correlates of non glandular stomach epithelial hyperplasia/inflammation/ulceration in stomach and lymphoid depletion in thymus were observed in the above groups.


HISTOPATHOLOGY (PARENTAL ANIMALS):
In stomach, the primary local lesion observed in the non glandular region of 600 mg/kgBwt/day dose group males and females and
150 mg/kgBwt/day dose group females was epithelial hyperplasia (minimal to moderate degree in severity) along with inflammation in a few rats. A
single incidence of non glandular stomach ulceration was also observed in 600 mg/kgBwt/day dose group females.

The incidences of the stomach lesions were higher in the females when compared to males. All the test item related stomach lesions reversed at
the end of recovery period.
In thymus, the incidences of lymphoid depletion observed in 150 and 600 mg/kgBwt/day dose group females were considered test item related and found to be reversible at the end of recovery period.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: there were no adverse effects observed in any parameters noted at this dose level.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for reproductive/developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Hematology:

There were no test item related hematological changes observed in males and females.

Coagulation:

There were no test item related changes observed in the coagulation parameters in both males and females.

Clinical chemistry:

There were no test item related changes observed in the clinical chemistry

parameters in both males and females.

Applicant's summary and conclusion

Conclusions:
To summarize, oral administration of test item UNDECYLENOYL PHENYLALANINE” to Wistar rats at the dose levels 50 and 150 mg/kg Bwt/day had no effects on general health, body weights, food intake, pre-coital time, gestation length, mating and fertility parameters. Functional observations
did not reveal any test item related changes at all the tested doses in both sexes. The live birth and survival index was not altered by the treatment at the entire tested dose. The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical
chemistry parameters of both males and females. There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females. Gross examination of pups on lactation day 4 did not reveal any gross changes. There were no test item related gross and microscopic changes in both males and females at 50 mg/kg Bwt/day. At 150 mg/kg Bwt/day, grossly single incidence of small sized thymus and thickening of non-glandular stomach were observed in females associated with non-glandular epithelial hyperplasia
microscopically.
At the 600 mg/kg Bwt/day, the treatment had no effects on general health, food intake, pre-coital time, gestation length, mating and fertility
parameters. Functional observations did not reveal any test item related changes at all the tested doses in both sexes. The live birth and survival
index was not altered by the treatment at the entire tested dose. The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females. The treatment resulted in significantly lower the body
weights males. Organ weights and organ weights ratios show a minimal decrease in thymus weight in females. Grossly small sized thymus
associated with microscopic correlate of lymphoid depletion, thickening of non-glandular stomach associated with microscopic changes of
epithelial hyperplasia of non-glandular stomach/ulceration/inflammation were observed essentially in female groups. Gross examination of pups on lactation day 4 did not reveal any gross changes.
In the recovery group, no microscopic changes observed indicating the reversibility of the effects observed during the treatment period. The
gross and microscopic examination of reproductive organs did not reveal any test item related changes at all the doses tested.
No Observed Adverse Effect Level :
The “No Observed Adverse Effect Level (NOAEL)” for systemic toxicity in parental rats is considered to be 50 mg/kg Bwt/day as there were no
adverse effects observed in any parameters.
The “No Observed Adverse Effect Level (NOAEL)” for reproductive/developmental toxicity is considered to be 600 mg/kg Bwt/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 and also to provide initial information on possible effects on male and female reproductive performance such as gonadal functions, mating behaviour, conception, development of conceptus and parturition.

The identity of the test item was provided by the Sponsor as a Certificate of Analysis (CoA). The authenticity of the test item was not done at the test facility. The stability and homogeneity of test item in the vehicle was carried out separately under Advinus Study No.G8470. Based on the results, the test item was found to be homogeneous and stable for up to 6 hours at 1 and 100 mg/mL

concentrations when stored at room temperature.

The test item was weighed and suspended in vehicle i.e., 0.5% Carboxymethyl cellulose with 0.1% Tween 80 and administered to rats at the graduated dose levels of 50, 150 and 600 mg/kg/day for 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 (0.5%

Carboxymethyl cellulose with 0.1% Tween 80) alone. The dose volume administered was 10 mL/kg body weight. 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 test item was administered once daily to specific group of rats prior to the mating period, during mating

period and during post mating period (for males), during pregnancy and up to lactation day 4 (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 day of sacrifice of the first littered animals.

All animals were observed for clinical signs, physical abnormalities and mortality. The body weight and food consumption were measured at periodic intervals. The functional observation battery was done shortly before sacrifice for 5 males and 5 females randomly selected from each group. For recovery groups functional observation battery was done prior to sacrifice. Laboratory

investigations such as haematology and clinical chemistry were performed for 5 males and 5 females randomly selected from each group at the end of the premating period for main groups and at the end of recovery period from all animals for recovery groups. The animals were subjected to detailed necropsy at sacrifice after overnight fasting and study plan specified tissues were collected. Tissues collected from randomly selected 5 males and 5 females in the control nd high dose groups were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis. All gross lesions were examined in all the groups. The stomach from males, stomach, thymus and adrenals from

females in the lower dose groups and recovery groups were examined as suspected test item related changes were observed in the high dose groups (G4).

Under experimental conditions described in the Material and Method section, the following results were obtained:

- No clinical signs or mortality was observed during the course of the study.

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

- The treatment resulted in decreased mean body weights at 600 mg/kg Bwt/day dose in males.The body weights and food consumption were unaffected during gestation and lactation periods at all the doses tested.

- Treatment had no effect on pre-coital time or gestation 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 Day 4 survival index was not altered by the treatment at all 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.

- There were no test item related changes in the terminal body weights. A minimal decrease in thymus weights were observed in females, at 600 mg/kgBwt/day dose, associated with small sized thymus grossly and lymphoid depletion microscopically. An isolated incidence of small sized thymus was also observed in 150 mg/kgBwt/day dose group females. At 600 mg/kgBwt/day dose group, thickening of non-glandular stomach was observed grossly.in females and, even less, in males, associated with epithelial hyperplasia of non-glandular stomach/ulceration/inflammation microscopically. In females, the gross lesion of non-glandular stomach thickening and microscopic change of non-glandular epithelial hyperplasia in stomach were also observed, to a lesser occurrence, at

150 mg/kgBwt/day dose group.

The gross examination of pups on lactation day 4 did not reveal any test item related changes.

No Observed Adverse Effect Level:

- The “No Observed Adverse Effect Level (NOAEL)” for systemic toxicity in parental rats is considered to be 50 mg/kg Bwt/day.

- The “No Observed Adverse Effect Level (NOAEL)” for reproductive and developmental toxicity is considered to be 600 mg/kg Bwt/day