2-(undec-10-enoylamino)-3-phenylpropanoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 for cross-reference:

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

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

UNDECYLENOYL PHENYLALANINE

IUPAC Name:

UNDECYLENOYL PHENYLALANINE

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 or test system 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 oral 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

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
other: nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
other: nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
600 mg/kg/day
Basis:
other: 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

Positive control:

No positive control

Examinations

Observations and examinations performed and frequency:

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:

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 males and 5 females of main group animals and at the end of recovery period all animals
- Parameters checked in table (page 28) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Animals fasted: Yes
- How many animals: 5 males and 5 females of main group animals and at the end of recovery period all animals
- Parameters checked in table (page 29) were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 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.
- Dose groups that were examined: Each
- Battery of functions tested: sensory activity / grip strength / motor activity / Landing Hindlimbs Footsplay

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality 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):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

An apparent decrease in the thymus weights (absolute) was observed in 600 mg/kgBwt/day dose group females

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

non-glandular stomach thickening; non-glandular stomach lesions were; small sized thymus

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

non glandular stomach ulceration; lymphoid depletion in thymus

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

epithelial hyperplasia in stomach

Details on results:

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

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.

Target system / organ toxicity

Critical effects observed:

not specified

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:

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.