trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline

Toxicity to reproduction

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:

2012-04-20 to 2012-12-31

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:

2012

Report date:

2012

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

Principles of method if other than guideline:

Nil

GLP compliance:

yes

Remarks:

OECD Guideline No. 422

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline

EC Number:

255-490-0

EC Name:

trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline

Cas Number:

41672-81-5

Molecular formula:

C37H69NO5

IUPAC Name:

1-palmitoyl-4-(palmitoyloxy)-L-proline

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline
- Molecular formula (if other than submission substance): NA
- Molecular weight (if other than submission substance): NA
- Smiles notation (if other than submission substance): NA
- InChl (if other than submission substance): NA
- Structural formula attached as image file (if other than submission substance): NA
- Substance type: NA
- Physical state: White powder
- Analytical purity: Consider as 100% (dry extract)
- Impurities (identity and concentrations): NA
- Composition of test material, percentage of components: NA
- Isomers composition: NA
- Purity test date: 2/04/2011
- Lot/batch No.: 1105500007
- Expiration date of the lot/batch: February 23rd 2014
- Radiochemical purity (if radiolabelling): NA
- Specific activity (if radiolabelling): NA
- Locations of the label (if radiolabelling): NA
- Expiration date of radiochemical substance (if radiolabelling): NA
- Stability under test conditions: NA
- Storage condition of test material: Ambient (+ 15 to +25 °C)
- Other: Nil

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: 11 -12 weeks
- Weight at study initiation: Males: 241 to 312g; Females: 171 to 223g
- 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):21 to 25°C
- Humidity (%): 65 to 68 %
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From:20 April 2012 To: 23 June 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Following procedure was followed when 100 mL of dose formulation prepared:
The quantities of 1000, 3000 and 10000 mg of test item was weighed on a separate aluminum foil and carefully transferred into separate mortor. Small aliquot of vehicle [0.5%w/v sodium salt of carboxymethyl cellulose (medium viscosity) with 0.1 % 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 10, 30 and 100 mg/mL for the G2, G3 and G4/G4R groups, respectively.


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):0.5% (w/v) Sodium Carboxymethyl cellulose (medium viscosity) with 0.1% (w/v) Tween® 80 in Milli-Q water was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study (study no. G8202). Indeed, this vehicle is commonly used for iterative studies, moreover the test item is well solubilized in this matrix. Further the historical data was also available with this vehicle.
- Concentration in vehicle: 0.5 %
- Amount of vehicle (if gavage): 10 mL/Kg body weight
- Lot/batch no. (if required): 039K0149
- Purity: NA

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 test item concentration (calculated based on determined concentration of DPHP as tracer) analysis, the prepared dose suspension was sampled in duplicate sets from each dose levels (two samples were drawn from each of the top, middle and bottom layers) on Day 1 and during 2nd month of the treatment period. Similarly, one sample from middle layer was drawn from vehicle control. One set of samples was sent to Analytical R&D of Advinus Therapeutics Limited, for analysis and the other set were stored in the refrigerator condition. The sample analysis was done as per the analytical method validated under Advinus Study No.: G8201.

On Day 1, samples corresponding to 10 mg/mL and 30 mg/mL were marginally outside the acceptance criteria of ±15%. Hence back-up samples were analysed. The results indicated that the test item concentrations in solution were within the permissible limits of ± 15 %.from the nominal concentrations.

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: 13 - 14 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 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 100 (G2), 300 (G3) and 1000 (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.G8202) 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:

Nil

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.?] were included.

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:

OTHER: Nil

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:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): No

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): No

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Not done

REPRODUCTIVE FUNCTION: No effects

REPRODUCTIVE FUNCTION: Not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): No effects

ORGAN WEIGHTS (PARENTAL ANIMALS) : No effects

GROSS PATHOLOGY (PARENTAL ANIMALS): No effects

HISTOPATHOLOGY (PARENTAL ANIMALS): No effects

OTHER FINDINGS (PARENTAL ANIMALS): Nil

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effect noted

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

Details on results (F1)

VIABILITY (OFFSPRING) : No effects

CLINICAL SIGNS (OFFSPRING): No effects

BODY WEIGHT (OFFSPRING): No effects

SEXUAL MATURATION (OFFSPRING): Not examined

ORGAN WEIGHTS (OFFSPRING): Not examined

GROSS PATHOLOGY (OFFSPRING): No effects

HISTOPATHOLOGY (OFFSPRING): Not examined

OTHER FINDINGS (OFFSPRING): Nil

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No significant effect noted

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Nil

Applicant's summary and conclusion

Conclusions:

To summarize, oral administration of test item “ trans-1-(1-oxohexadecyl)-4-[(1-oxohexadecyl)oxy]-L-proline” to Wistar rats at the dose levels 100, 300 and 1000 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

No Observed Adverse Effect Level (NOAEL):

As there were no adverse effects observed in any parameters up to the highest dose tested, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg Bwt/day.

Executive summary:

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 body weights and food consumption were unaffected by the treatment at all the doses tested.

The body weights and food consumption were unaffected during gestation and lactation periods at all the doses tested.

There were no treatment-related changes in the hematology, coagulation and clinical chemistry parameters.

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, 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.

No Observed Adverse Effect Level (NOAEL): As there were no adverse effects observed in any parameters up to the highest dose tested, the No Observed Adverse Effect Level (NOAEL) is considered to be 1000 mg/kg Bwt/day.