N-(2-ethoxyphenyl)-N'-(4-isododecylphenyl)oxamide

Endpoint summary

Description of key information

Daily oral (gavage) administration of test item “Hostavin 3206 LIQ (Impoverished Xylene)” to Wistar rats at the dose levels 100, 300 and 1000 mg/kg Bwt/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) 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. The survival indices were not altered by the treatment. The test item administration did not reveal any changes in the hematology, coagulation and clinical chemistry parameters. 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 LD 13 did not reveal any gross changes. There were no microscopic changes observed in both males and females. Further, the male and female reproductive organs did not reveal any changes. The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test itemadministration.

No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of parental rats and pups.

The No Observed Adverse Effect Level (NOAEL) of is considered to be 1000 mg/kg Bwt/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose 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

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

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Department of Safety Assessment Advinus Therapeutics Limited Bengaluru 560 058, India

- Age at study initiation: 14-16 weeks
- Weight at study initiation: Males: 394 to 526 g Females: 231 to 282 g

- Fasting period before study:
- Housing:
- Use of restrainers for preventing ingestion (if dermal): yes

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, Wi 53744-4420 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 Limted., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment. During the acclimatization period, animals were observed once daily for any abnormalities. Only the animals that are determined by the veterinarian to suitable for use were assigned to this study. Female rats used in this study were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 and 24°C and relative humidity between 59 and 68 %. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12 - 15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

- Temperature (°C): 21 and 24°C.
- Humidity (%): 59 and 68 %.
- Air changes (per hr):12 - 15 changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 29 March 2016 To: 04 July 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily prior to start of the treatment.
Required quantities of the test item were weighed in to a pre-calibrated beaker* and small volume of corn oil was added. The resulting pre-mix was warmed. This pre-mix was allowed to thaw to room temperature and the volume was made up with the vehicle to attain desired concentrations of 20, 60 and 200 mg/mL for the G2, G3 and G4/G4R groups, respectively.

Pre-calibration of the beaker to desired volume: Milli-Q water was measured in a graduated cylinder to the final volume of the batch size (70 mL). The measured water was transferred into a clean beaker (to be pre-calibrated) and upper and lower meniscus of water was marked on the beaker using a marker. Once these lines were marked, the water was discarded and the beaker was dried. The volume was made up to the upper meniscus when preparing the dose formulations.

Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring using a magnetic stirrer.

The volume of dose formulation prepared was varied depending on the requirement and/or body weights of the rats recorded during experimental period.


VEHICLE
Corn oil was used as vehicle for dose formulation preparation as the same vehicle was used in the dose range finding toxicity study (Study No. N2825) with the same test item.

Details of components used for vehicle preparation are as follows:

- Name of vehicle: Corn oil
- Manu-factured by: Sigma
- Lot no.: MKBV2080V
- Date of receipt: 19.10.2015
- Date of expiry: 18.10.2020



Name of vehicle Manu-factured by Lot no. Date of receipt Date of expiry
Corn oil Sigma MKBV2080V 19.10.2015 18.10.2020

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 analysed in-house. 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. G11306. 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.
Formulations were considered acceptable as mean results are within ± 15 % of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.

Duration of treatment / 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 during the post mating period until sacrifice.
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 LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 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 dose volume administered for each rat was 5 mL/kg Bwt throughout the study except for rat number Rt3330 (G2F) wherein 5.1 mL was administered from experimental Day 22 to 28. 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 5 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.

Frequency of 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 during the post mating period until sacrifice.

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 LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 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 dose volume administered for each rat was 5 mL/kg Bwt throughout the study except for rat number Rt3330 (G2F) wherein 5.1 mL was administered from experimental Day 22 to 28. 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 5 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.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 Males and 10 Females per each main groups. 5 Males and 5 Females for recovery group.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: G1 '0' mg/kg Bwt/day; G2 '100' mg/kg Bwt/day; G3 '300' mg/kg Bwt/day; G4 '1000' mg/kg Bwt/day; G1R '0' mg/kg Bwt/day; G4R '1000' mg/kg Bwt/day

- Rationale for animal assignment : Grouping was done by the method of body weight stratification and distribution. On the day of randomization, based on the given temporary animal identification number, each animal with normal oestrous cyclicity (4-5 day cycle) was weighed and the corresponding body weight was recorded. The data was transferred to EDP (Electronic Data Processing) for data input (temporary identification number and body weight) into an excel spread sheet. The body weight recorded was stratified in ascending order.

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 (FOB) Tests
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, observations during removal of animal from home cage and handling, open filed observation, functional tests, motor activity, sensory reactivity measurements, landing hindlimb footsplay, grip performance, physiological obsevations)

- 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 GD 0, 7, 14 and 20 and on 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.

- Oestrous Cycle Evaluation
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. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating period to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. The overall pattern of each female was characterized as regular cycling (having recurring 4– 5 day cycles) and irregularly cycling (having cycles with a period of diestrus longer than 3 days or a period of estrus more or less than 2 days). Incomplete cycles (having prolonged periods of either estrus or diestrus) were not included in calculating the mean cycle length.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

- Blood Collection
At the end of the pre-mating period (Day 16), randomly selected 5 males and 5 females of each main group and at the end of recovery period from all rats were fasted overnight (water allowed) and blood was collected by retro-orbital plexus puncture under isoflurane anaesthesia. Approximately 3 mL of blood was collected.
A. Haematology:
red blood corpuscles, haemoglobin, haematocrit, mean corpuscular volume, mean corpsuclar haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes, white blood corpuscles, differential leukocyte count, platelets, prothrombin time, activated partial thrmboplatin time

B. Clinical chemistry:
alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, albumin/globulin ratio, total bile acids, blood urea nitrogen, calcium, chloride, creatinine, gamma glutamyl transferase, glucose, globulin, inorganic phosphorus, potassium, sodium, total bilirubin, total cholesterol, total plasma protein, triglycerides

- Urinaliysis
Urine was collected at the end of the pre-mating period, from randomly selected 5 males and 5 females of main groups and at the end of recovery period from all rats, in urine collection tubes. For urine collection, each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis.
(specific gravity, nitrite, pH, proteins, glucose, ketone bodies, urobilinogen, bilirubin, leukocytes, erythrocytes, colour, gravity, volume)

- Hormone Analysis
Blood samples were collected and serum was separated as per the following schedule for the determination of total T4 and TSH:
• Two pups per litter on LD 4 after birth
• All dams and at least two pups per litter on LD 13
• All adult males, prior to sacrifice.
The blood from pups was pooled by litter for thyroid hormone analyses. Pups were lightly anaesthetized 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.

- Thyroid Profile Hormones
The following thyroid hormones were estimated by Enzyme linked immuno sorbent assay (ELISA) method for the samples as stated under section 8.9.6 using BIO-RAD microplate washer and BIO-RAD model 680 reader.
Serial No. Parameters Abbreviations Unitsc
1 Rodent Thyroid Stimulating Hormone TSH ng/mL
2 Rodent Thyroxin T4 ng/mL
c: expansion of unit: ng/mL: nano grams/ per milli litre
The kits manufactured by Endocrine Technologies Inc., USA were used for the assay.

Sacrifice and pathology:

- Necropsy
All adult animals and pups LD 13; including dead pups) were examined macroscopically for any structural abnormalities/pathological changes and findings were recorded. The adult animals killed at term were fasted overnight (water allowed), exsanguinated under isoflurane anaesthesia and weighed prior

- Histopathology
Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland collected from LD 13 pups from all the groups were also evaluated. All gross lesions were examined from all the groups.
The tissues were processed for routine paraffin embedding and 4 - 5 micron thickness sections were stained with Mayer’s Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 μm thickness and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis.
Unused tissues will be archived.
(axillary lymph nodes, sdrenals, brain, bone marrow smear, cecum, colon, duodenum, eyes (with optic nerve)femur with marrow, heart, ileum with peyer's patches, jejunum, kidneys, liver, lungs, mammary gland, mandibular lymph nodes, mesenteric lymph nodes, pituitary, rectum, sciatic nerve, skeletal muscle, spinal cord, spleen, sternum with marrow, stomach, thymus,trachea, urinary bladder)

Statistics:

STATISTICAL ANALYSES
ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data was entered retrospectively in ProvantisTM) and Clinical Chemistry data was analysed using Provantis built- in statistical tests. 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 neurological observations (neuromuscular observation/body temperature/body weights), body weight, net weight gain, food consumption, oestrous cycle length, hormone levels, ano-genital distance, body weights, ano-genital index, mean number and weight of pups, 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 heteroschedastic) data was transformed, before ANOVA was 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. In case of recovery groups, data was analysed using Two sample t-test. Comparison of means between treatment recovery group(s) and vehicle control recovery group was performed.
el.
Statistically significant differences (P<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed at any of the doses tested.
However, the clinical sign of sparse hair loss was observed in one male rat in the control recovery and three female rats in the high dose recovery groups. The observation was considered as spontaneous finding and not related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality was observed at any of the doses tested. However, an incidence of one female rat (Rt3272) in the vehicle control group was died after blood sample collection during pre-mating period, could be due to over anaesthesia.

Body weight and weight changes:

no effects observed

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:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adeverse effects observed up to limit dose

Key result

Critical effects observed:

no

Conclusions:

Based on the results of this OECD 422 study the NOAEL for Hostvin 3206 was derived at 1000 mg/kg bw/d (highest dose tested):

Executive summary:

To summarize, daily oral (gavage) administration of test item “Hostavin 3206 LIQ (Impoverished Xylene)” to Wistar rats at the dose levels 100, 300 and 1000 mg/kg Bwt/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) 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. The survival indices were not altered by the treatment. The test item administration did not reveal any changes in the hematology, coagulation and clinical chemistry parameters. 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 LD 13 did not reveal any gross changes. There were no microscopic changes observed in both males and females. Further, the male and female reproductive organs did not reveal any changes. The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test itemadministration.

No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of parental rats and pups.

The No Observed Adverse Effect Level (NOAEL) of is considered to be 1000 mg/kg Bwt/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restriction

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Due to the NOAEL of 1000 mg/kg bw/day in an OECD 422 repeated dose/reproduction/developmental toxicity screening test in rats the registration substance does not have to be classified regarding systemic and target organ toxicity after repeated exposure according to the criteria laid down in the EU Classification Labelling and Packaging Regulation (1272/2008/EC).