N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-16 to 2022-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-16 to 2022-04-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Conditions: The animal room was air-conditioned with adequate (above 10) air changes per hour. The room was continuously monitored for temperature and relative humidity. The ranges for room temperature and relative humidity were 20.2 to 22.3°C and 57 to 65 %, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.
Accommodation: Initially (acclimatization and randomization period), all animals were housed in groups of two to three per polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with paddy husk bedding. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited. After randomization, males and females were individually in grouped. During the mating phase, animals were housed on one male: one female basis within each dose group.
Enrichment: Enrichment were provided for singly housed animals. After successful mating, the females were returned to their original cages and housed individually during gestation and lactation.
Diet: Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot No. 2014C-091720MA) from ENVIGO was provided ad libitum. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.
Water: UV Purified water will be provided ad libitum. Results of bacteriological, chemical and contaminant analyses will be archived at RCC Laboratories India Private Limited.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Dose volume: 10 mL/kg body weight
Corn oil is selected as vehicle based on preliminary solubility testing which was performed at the test facility before the study initiation date.
The test item was formulated in vehicle. The dose formulations were prepared shortly before each dosing. The test item was weighed into a pre-weighed glass beaker on an appropriate weighting balance. 5-10 ml of vehicle was added to mortar, this was triturated using pestle. This was transferred to measuring cylinder and final volume was made in the measuring cylinder. This was transferred back to beaker. Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Due to the insolubility in organic sovents an analytical verification by LC or GC was not suitable. The assay was carried out by gravimetric method. Different concentrations of test item in corn oil was extracted using suitable solvent and the residue was dried and weighed. Appropriate volume of test item in corn oil was taken in a 15 mL centrifuge tube, followed by addition of acetone up to 10 ml volurne and capped with screw lid air tight. The contents were mixed weil using vortex-mixer. After thorough mixing the sarne was centrifuged at 3000 rpm for 5 minutes. The corn oil being miscible with acetone will form the supematant layer, while the test item settles at the cone of the centrifuge tube. The supernatant was discarded, by carful decanting without disturbance to the test itern settled in the cone of centrifuge tube. Additionally three such extractions with fresh acetone on the test item settled in the cone of centrifuge was carried out. During every extraction the supematant layer was decanted carefully and discarded. The remaining test item in the centrifuge tube was finally washed with n-Hexane as per the above extraction procedure to ensure removal of trace amount of oil. The remaining test item in the cone of the centrifuge tube was quantitatively transferred to pre-weighed petriplate and dried at 70°C for lhr. After one hour the petriplate was transferred to desiccator to cool to room temperature. After cooling, the petriplate with the test item was weighed and recorded. The method developed and validated at RCC Laboratories was found to be precise and accurate for the determination of Bisamid in corn oil matrix. The results arrived from the data generated during dose verification phase reveals that the test item in corn oil was matching with the nominal concentration with minimum of 86.67% and maximum of 97.53 %.

Duration of treatment / exposure:

Males were administered with test item up to 42 days; Fernales were administered with test item during premating, mating, gestation periods and up to day 13 post-partum

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

500 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:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose level selection: Dose range finding experiment was carried out before the main experiment to confirm the high dose level for test item. A total of three groups (Low, Mid and High) consisting of 4 Male and 4 Female rats was treated for a period of two week at dose levels of 100, 300 and 1000 mg/kg body weight. All animals were observed for mortality/morbidity, clinical signs of toxicity, Body weight (day 0, 7 and end of treatment). At the end of two week all animals were sent for necropsy for gross pathological examinations. A dose level for main study was selected based on dose range finding experiment observation and results.

Positive control:

no

Observations and examinations performed and frequency:

Mortality/ Viability : Twice daily

Clinical Signs : Once daily/Twice daily on initial 3 days after treatment, gestation, lactation period and once daily thereafter
(Daily cage-side clinical observations)
- Acclimatization Period
- Treatment Period

Feed Consumption : Males: Once weekly until termination (Except during Mating) Females: Premating: Once weekly; Gestation: Day 7, Day 14, Day 20; Lactation: Day 1, Day 4 , Day 13
- Treatment Period

Body Weights : Once Weekly Males: Individual body weights were recorded on first day of dosing, weekly thereafter and at termination. Females: Premating : Once weekly; Gestation: Day 0, 7, 14; 20; Lactation: Day 1, 4, 13
- Acclimatization Period
- Treatment Period

Detailed clinical Observation : Treatment Period: Males & Females: Before dosing: Once weekly

Sensory reactivity, Grip Strength : Prior to termination
and Motor Activity

Reproductive indicies
The following data was evaluated
 Mating performance and fertility (Pre-coital interval, mating index and pregnancy index)
 Gestation and parturition Data (Gestation length and parturition index)
 Litter Responses (Pre implantation loss, live/birth index and sex ratio)

animal selection for repeated toxicity sreening
Five males and five females were selected randomly from each test and control group prior to termination (Day before the terminal sacrifice)

Sacrifice and pathology:

necropsy: All animals found dead or killed were subjected to complete gross necropsy. Females which failed to achieve pregnancy or produce a litter were sacrificed on or after Day 26 post coitum. Dead pups were subjected to gross examination. Day 4 pups were sacrificed by thiopentone sodium and day 13 pups by CO2 asphyxiation and gross examination were performed. All the animals were subjected to gross examination. The male animals were sacrificed by CO2 asphyxiation on Day 43 and female animals were sacrificed Day 14 post-partum by CO2 asphyxiation. The uteri of females were examined for the presence and number of implantation sites. All animals were weighed and sent to the necropsy. Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected from; Group 1, 2, 3 and 4 (Randomly selected five males and five females from each sex/ group) animals at necropsy and fixed in 10% Neutral Buffered Formalin solution (NBF) except testes, epididymides and eyes which were fixed in modified Davidson’s fixative for 24 hours and transferred to 10% NBF. From all adult males and females and one male and one female day 13 pup from each litter, thyroid glands were preserved. All organ and tissue samples, as defined under histopathology, were processed, embedded and cut at an approximate thickness of 3 to 5 micrometers, and stained with hematoxylin and eosin. Full histopathology was performed on the preserved organs and tissues of animals of Group 1 and 4 (Randomly selected five males and five females from each sex/ group). All gross lesions were examined microscopically. Microscopic examination of slides was done by Study Pathologist.

Statistics:

The following statistical methods were used to analyze the body weight, feed consumption, hematological, biochemical parameters and organ weight data, Pre-coital interval, gestation length, litter size and litter weights, AGD, sex ratio and implantation sites, implantation losses, viability indices, offspring body weight and body weight change. Statistical analysis of pup body weight was based on individual pup data, taking litter size into account.
• Data was summarized in tabular form. Statistical analysis was performed using Stat Plus program
• The continuous data was checked for Normality with Shapiro-Wilk test and subjected to perform Analysis of Variance (ANOVA) to compare the difference between treated and control groups.
• Discontinuous data was subjected to non-parametric tests, for two groups Mann-Whitney U-Test and for more than two groups kruskal-wallis ANOVA was used.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male during treatment period significant increase in A/G and decrease in GLB was noticed in high dose (G4-1000 mg/kg body weight) group compared to control (G1-0 mg/kg body weight. In female significant increase Cholesterol in high dose (G4-1000 mg/kg body weight) was noticed compared to control group (G1-0 mg/kg body weight). The significance changes observed in biochemical parameters are non-dose dependent and no biological significant and hence could not be attributed to treatment related adverse effect.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males, absolute and relative organ weight of kidneys and thymus belonging to intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) group was significantly decreased when compared with G1. Absolute and relative organ weight of kidneys belonging to G2-250 mg/kg body weight) was significantly decreased as compared to G1. In males, absolute and relative organ weights of epididymides and prostate, seminal vesicle with coagulating glands belonging to G2, G3 and G4 was significantly decreased as compared to G1. The all above significances can be considered as incidental as there were no changes in gross and histopathology of the organs.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

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: highest dose tested

Key result

Critical effects observed:

no

Conclusions:

The administration of Bisamid to Wistar rats by oral gavage at dose levels of 250, 500 and 1000 mg/kg body weight/day revealed no treatment-related clinical signs, changes in the body weights or feed consumption. No treatment-related effects on reproduction/development such as mating index, fertility index, gestation length, post-natal loss, sex ratio and offspring growth and development. Macroscopic examination revealed no test item related findings in any of the animals of 250, 500 and 1000 mg/kg body weight body weight as group. No abnormality was observed attributable to test item in pups from all treatment groups. Microscopic examination revealed no abnormality attributable to test item - Bisamid at the highest dose tested i.e. 1000 mg/kg body weight. No Observed Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test: 1000 mg/kg body weight.

Executive summary:

The test item Bisamid, formulated in corn oil was administered by oral gavage to three groups, each of twelve male and twelve female Wistar rats, up to 42 days for males were treated, for two weeks premating phase, up to two weeks mating and post-mating. Females two weeks premating phase, up to two weeks mating, three weeks gestation and 13 days Iactation for females at dose levels of 250, 500 and 1000 mg/kg body weight/day. A control group of twelve males and twelve females were dosed with vehicle control (corn oil). The following observation and examinations were evaluated: Clinical signs (daily), body weights and feed consumption (once weekly except during mating). Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring body weights. Five male and five female animals selected for repeated dose toxicity screening. Males were sacrificed on day 43, followed by the sacrifice of all females on day 14 post-partum. Pups were sacrificed on Day 13 postpartum. Macroscopy at termination was performed for all animals. All males and females were evaluated for Hematology, Clinical biochemistry and urine analysis at the end of treatment period. Male and female animals were screened for T4 and TSH parameters from each dose group. Selected offspring were also screened for T4 and TSH parameters from each dose group. Testes, epididymides, seminal vesicle and prostate of all adult male; uterus, ovaries of all female animals and following organ weights were recorded on scheduled dates of necropsy from five male and five female animals selected for repeated dose toxicity screening. Wet weights were taken immediately after dissection to avoid drying. From all adult males on day 43 and females on day l4 postnatal and two pups from day 13 postnatal from each litter and thyroid weights was determined after fixation. All organ and tissue samples as defined under histopathology were processed, embedded and cut at an approximate to thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. Full histopathology was performed on the preserved argans and tissues of animals of group 1 and 4 (Randomly selected five males and five females from each sex/ group). All gross lesions were examined microscopically.

Results

Analytics:

Analysis of stability, homogeneity and dose concentration of the prepared dose formulations revealed acceptable levels. The dose formulation analytical report provided is appended to the study report.

Mortality:

There were no mortalities observed in control group (0 mg/kg body weight), low dose group (250 mg/kg body weight), intermediate dose group (500 mg/kg body weight) and high dose group (1000 mg/kg body weight) animals in premating, mating, gestation and lactation period. However, cannibalism was observed in one dam (Dam No. 021) of control dose group on day 2 lactation period. In low dose group (250 mg/kg body weight), cannibalism was observed in one dam on lactation day 1 in dam No, 037 and each one female in dam 42 and 48. Cannibalism was observed in high dose group (1000 mg/kg body weight) three female pups and one male on lactation day 3 in dam No, 091. The mortalities observed in Pups could not be attributed to test item which are common incidental findings in strain and species.

Clinical signs:

No clinical signs were observed in the control group (G1- 0 mg/kg body weight), low dose group (G2 - 250 mg/kg body weight), intermediate dose group (G3-500 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) in both male and female animals.

Detailed Clinical Examination and Functional observation battery:

No abnormalities were observed in all the animals in low (G2- 250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) in both the sexes when compared with control animals. Functional observation battery parameters like grip strength (Forelimbs and hind limbs), Rotarod and IR Actimeter did not show any treatment related significant changes in all the treated groups when compared with control group.

Body weight:

Body weight and body weight gain of male and female of all treated animals were comparable with the control group animals.

Feed Consumption:

The significant changes were observed in feed consurnption in low dose (G2-250 mg/kg intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal and could not be attributed to test item administration.

Clinical Patbological Investigations:

The significance differences observed in hematology and clinical biochemistry parameters could not be attributed to treatment due to marginal increase or decrease in individual control values and no any biological significant. No treatment related significant differences were observed urinalysis parameter in all treated groups in both the sexes. No effects were detected on mating performance. All the female animals showed positive evidence mating. No significant difference was observed in the pre-coital interval of female animals between control and dose groups.

Reproductive indices:

Oestrous cyclicity was checked for all the female animals. Regular oestrous cycle was observed in all the animals of different groups in the study. Mating index for all the groups was 100% (as confirmed by presence of sperm in the vaginal smear). The two Dams from control (Gl-0 mg/kg body weight), two dams in low (G2-250 mg/kg body weight), intermediate dose (G3-500 mg/kg body weight) and one dam high dose (G4-1000 mg/kg body weight) was observed non pregnant. The pregnancy index for control (G 1- 0 mg/kg body weight), low (G2-250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high (G4 -1000 mg/kg body weight) was found to be 83.33, 83.33, 83.33 and 91.67, respectively. Of the litters delivered, in all the treatment groups, litter size at birth and on Day 1 and 4 post-partum were comparable to controls. No significant difference in the number of live births was noted between control and treated groups. There were no effects in fertility of treated animals.There were no differences in gestation lengths. The gestation length for treated females was comparable to controls. All animals showed gestation lengths between 21 to 24 days.The parturition index was 91.67 % in high dose group and whereas in control, low and intermediate dose it was observed 83.33 %.No significance changes were observed in post natal losses in the entire treated group in both sexes, sex ratio was calculated as percent (%) male. No significant difference in sex ratio was observed in any of the treated groups when compared with vehicle control. Offspring body weight on day 0, 4 and 13 were recorded. The significant changes were observed in Offspring body weight in intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal on day 0, 4 and 13 in male. The significant changes were observed in Offspring body weight in intermediate (G3-500 mg/kg body weight) are marginal on day 4 in female and could not be attributed to test item administration. No significant differences in the Anogenital distances (AGD) between control and treated male animals were observed. In females, the significant changes were observed in AGD in low (G2-250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal on day 4. In combined for both sexes, the significant changes were observed in AGD in low (G2-250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal on day 4.

Terminal fasting body weights and organ weights:

Terminal fasting body weights, absolute and relative organ weights in males and females were comparable between control and high dose groups. Selected animals for organ collection: In males, absolute and relative organ weight of Kidneys and thymus belanging to intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) group was significantly decreased when compared with G 1. Absolute and relative organ weight of kidneys belonging to G2-250 mg/kg body weight) was significantly decreased as compared to G1. No significance was observed in terminal body weight, absolute and relative organ weight of randomly selected females when compared to control G1. Remaining animals: In males, absolute and relative organ weights of epididymides and prostate, seminal vesicle with coagulating glands belonging to G2, G3 and G4 was significantly decreased as compared to G1.Pups: Absolute and relative thyroid weight of pups belonging from G3 was significantly increased when compared with G1. No significance was observed in terminal body weight, absolute and relative organ weight of remaining females when compared to control G1. The all above significances can be considered as incidentalas there were no changes in gross and histopathology of the organs.

Macroscopig fndings

There were no treatment related macroscopic findings in males and females and in pups. Males: Two incidences of bilateral small sized testes in group 2 (Animal number 035 and 036) and one incidence in group 4 (Animal number 077) was observed. Microscopically slight to moderate degenerative changes in the seminiferous tubules of testes were observed in animal number 035 and 036. Marked seminiferous tubular atrophy was observed in animal number 077. Single incidence of bilateral small sized epididymides in group 4 (Animal number 077) showed marked oligospermia. One incidence of enlarged spleen in group 2 (Animal number 058) revealed extramedullary haematopoiesis on microscopic exarnination. Females: Single incidence of dilated uterus observed (Animal No.018) and was microscopically confirmed to be dilatation of the uterine lumen. This was considered to be a physiological change.

Microscopic findidings:

There were no treatment related microscopic findings in testes and epididymides in males and in ovaries in females at high dose. Males: In control group, two incidences of vacuolation of hepatocytes (Animal number 011 and 012) and single incidence of bile duct hyperplasia (Animal number 012) in liver was observed. Infiltration of mononuclear cells in kidneys (Animal number 012) was observed. Lungs revealed two incidences of increased alveolar macrophages, one in control (Animal number 009) and one in high dose group animal (Animal number 075), severity of lesion was comparable. Single incidence of infiltration of mononuclear cells was observed (Animal number 010). One incidence of vacuolation of zona fasciculata in control (Animal number 001) and one in high dose group animal (Animal number 084) was observed. Cell debri was found in the lumen of seminiferous tubules of testes of two control group animals (Animal number 011 and 012). One incidence of single cell necrosis and oligospermia in epididymides of control group animal (Animal number 010) was observed. High dose group animals revealed two incidences of infiltration of mononuclear cells in prostate gland (Animal number 079 and 084). Single incidence of cyst and pars distalis of pituitary gland was absoerved in control group animal (Animal number 010).Females: Two incidences of presence of alveolar macrophages in lung of control group animals was observed (Animal number 021 and 022). Single incidence of microscopic findings observed in various organs in control and treatment groups were considered incidental. These changes were not considered adverse as the incidences and severity was low and there were no changes in the weight of these organs. There were no treatment related changes in other vital organs such as liver, kidneys, heart, brain and reproductive organs such as testes and ovaries.

Conclusion:

The administration of Bisamid to Wistar rats by oral gavage at dose levels of 250, 500 and 1000 mg/kg body weight/day within the scope of OECD 422 revealed no treatment-related clinical signs, changes in the body weights or feed consumption. No treatment-related effects on reproduction/development such as mating index, fertility index, gestation length, post-natal loss, sex ratio and offspring growth and development. Macroscopic examination revealed no test item related findings in any of the animals of 250, 500 and 1000 mg/kg body weight body weight as group. No abnormality was observed attributable to test item in pups from all treatment groups. Microscopic examination revealed no abnormality attributable to test item - Bisamid at the highest dose tested i.e. 1000 mg/kg body weight. No Observed Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test: 1000 mg/kg body weight.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

EC Number:

204-613-6

EC Name:

N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Cas Number:

123-26-2

Molecular formula:

C38H76N2O4

IUPAC Name:

12-hydroxy-N-[2-(12-hydroxyoctadecanamido)ethyl]octadecanamide

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Conditions: The animal room was air-conditioned with adequate (above 10) air changes per hour. The room was continuously monitored for tem¬pera¬ture and relative humidity. The ranges for room tem¬pera-ture and relative humidity were 20.2 to 22.3°C and 57 to 65 %, respectively. The animals were provided with a light cycle of 12 hours light and 12 hours dark.
Accommodation: Initially (acclimatization and randomization period), all animals were housed in groups of two to three per polycarbonate cages (approximate internal dimensions of 365 mm x 202 mm x 180 mm height) with paddy husk bedding. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited. After randomization, males and females were individually in grouped. During the mating phase, animals were housed on one male: one female basis within each dose group.
Enrichment: Enrichment were provided for singly housed animals. After successful mating, the females were returned to their original cages and housed individually during gestation and lactation.
Diet: Teklad Certified Global 14% Protein Rodent Maintenance Diet (Lot No. 2014C-091720MA) from ENVIGO was provided ad libitum. Results of analyses for contaminants will be archived at RCC Laboratories India Private Limited.
Water: UV Purified water will be provided ad libitum. Results of bacteriological, chemical and contaminant analyses will be archived at RCC Laboratories India Private Limited.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Dose volume: 10 mL/kg body weight
Corn oil is selected as vehicle based on preliminary solubility testing which was performed at the test facility before the study initiation date.
The test item was formulated in vehicle. The dose formulations were prepared shortly before each dosing. The test item was weighed into a pre-weighed glass beaker on an appropriate weighting balance. 5-10 ml of vehicle was added to mortar, this was triturated using pestle. This was transferred to measuring cylinder and final volume was made in the measuring cylinder. This was transferred back to beaker. Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

Details on mating procedure:

Animals were paired on a one male: one female basis within each dose group, for a maximum period of ten days. Each female was examined for the presence of a copulation plug in the vagina. The presence of sperm in the vaginal smear was taken as positive evidence of mating (Day 0 of gestation). Each pregnant female was observed twice a day for three days during gestation and lactation period. The following was recorded for each female:
i) Date of pairing
ii) Date of positive evidence of mating
iii) Date of observed parturition

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Due to the insolubility in organic sovents an analytical verification by LC or GC was not suitable. The assay was carried out by gravimetric method. Different concentrations of test item in corn oil was extracted using suitable solvent and the residue was dried and weighed. Appropriate volume of test item in corn oil was taken in a 15 mL centrifuge tube, followed by addition of acetone up to 10 ml volurne and capped with screw lid air tight. The contents were mixed weil using vortex-mixer. After thorough mixing the sarne was centrifuged at 3000 rpm for 5 minutes. The corn oil being miscible with acetone will form the supematant layer, while the test item settles at the cone of the centrifuge tube. The supernatant was discarded, by carful decanting without disturbance to the test itern settled in the cone of centrifuge tube. Additionally three such extractions with fresh acetone on the test item settled in the cone of centrifuge was carried out. During every extraction the supematant layer was decanted carefully and discarded. The remaining test item in the centrifuge tube was finally washed with n-Hexane as per the above extraction procedure to ensure removal of trace amount of oil. The remaining test item in the cone of the centrifuge tube was quantitatively transferred to pre-weighed petriplate and dried at 70°C for lhr. After one hour the petriplate was transferred to desiccator to cool to room temperature. After cooling, the petriplate with the test item was weighed and recorded. The method developed and validated at RCC Laboratories was found to be precise and accurate for the determination of Bisamid in corn oil matrix. The results arrived from the data generated during dose verification phase reveals that the test item in corn oil was matching with the nominal concentration with minimum of 86.67% and maximum of 97.53 %.

Duration of treatment / exposure:

Males were administered with test item up to 42 days; Fernales were administered with test item during premating, mating, gestation periods and up to day 13 post-partum

Frequency of treatment:

daily

Details on study schedule:

- Groups of twelve male and twelve female animals were treated daily at dose level of 250 (low dose), 500 (Intermediate dose) and 1000 (High dose) mg/kg body weight throughout the study. The day of dosing was designated as 1 of the study.
- On Day 15, animals were paired on a one male: one female base within each dose group for a period up to ten days
- Following positive evidence of mating the males were returned to their original cages and females were housed individually.
- Pregnant females were allowed to give birth and maintain their survived offspring until Day 13 post-partum.
- Each litter was examined as soon as possible after delivery to establish number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than corresponding control pups) and the presence of gross abnormalities
- Five males and five females were selected randomly from each test and control group prior to termination (after day 42 for males and day 13 post-partum for females) for general toxicity evaluations.
- Vaginal smears were examined on the day of necropsy to detemrine the stage of the oestrous cycle

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose level selection: Dose range finding experiment was carried out before the main experiment to confirm the high dose level for test item. A total of three groups (Low, Mid and High) consisting of 4 Male and 4 Female rats was treated for a period of two week at dose levels of 100, 300 and 1000 mg/kg body weight. All animals were observed for mortality/morbidity, clinical signs of toxicity, Body weight (day 0, 7 and end of treatment). At the end of two week all animals were sent for necropsy for gross pathological examinations. A dose level for main study was selected based on dose range finding experiment observation and results.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

Mortality/ Viability : Twice daily

Clinical Signs : Once daily/Twice daily on initial 3 days after treatment, gestation, lactation period and once daily thereafter
(Daily cage-side clinical observations)
- Acclimatization Period
- Treatment Period

Feed Consumption : Males: Once weekly until termination (Except during Mating) Females: Premating: Once weekly; Gestation: Day 7, Day 14, Day 20; Lactation: Day 1, Day 4 , Day 13
- Treatment Period

Body Weights : Once Weekly Males: Individual body weights were recorded on first day of dosing, weekly thereafter and at termination. Females: Premating : Once weekly; Gestation: Day 0, 7, 14; 20; Lactation: Day 1, 4, 13
- Acclimatization Period
- Treatment Period

Detailed clinical Observation : Treatment Period: Males & Females: Before dosing: Once weekly

Sensory reactivity, Grip Strength : Prior to termination
and Motor Activity

animal selection for repeated toxicity sreening
Five males and five females were selected randomly from each test and control group prior to termination (Day before the terminal sacrifice)

Oestrous cyclicity (parental animals):

Vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

The day of completion of parturition was the Day 0. The number of live and dead offspring was recorded on Day 1 post-partum. Offspring was individually identified within each litter by body marking on Day 1. For each litter the following parameters were recorded:
i) Number of offspring born
ii) Number of offspring alive was recorded daily and reported on days 1 and 4 post-partum
iii) Sex of offspring on day 1 post-partum
iv) Clinical condition of offspring from birth to day 13 post-partum
v) Individual offspring body weights on Days 1, 4 and day 13 post-partum
vi) The Anogenital distance of each pup was measured on day 4 post-partum. The anogenital distance was measured from the cranial edge of the anus to the base of genital tubercle.
vii) The number of nipples in male pups was counted on day 13 post-partum
viii) Gross anomalies

Postmortem examinations (parental animals):

necropsy: All animals found dead or killed were subjected to complete gross necropsy. Females which failed to achieve pregnancy or produce a litter were sacrificed on or after Day 26 post coitum. All the animals were subjected to gross examination. The male animals were sacrificed by CO2 asphyxiation on Day 43 and female animals were sacrificed Day 14 post-partum by CO2 asphyxiation. The uteri of females were examined for the presence and number of implantation sites. All animals were weighed and sent to the necropsy. Descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs were collected from; Group 1, 2, 3 and 4 (Randomly selected five males and five females from each sex/ group) animals at necropsy and fixed in 10% Neutral Buffered Formalin solution (NBF) except testes, epididymides and eyes which were fixed in modified Davidson’s fixative for 24 hours and transferred to 10% NBF. From all adult males and females and one male and one female day 13 pup from each litter, thyroid glands were preserved. All organ and tissue samples, as defined under histopathology, were processed, embedded and cut at an approximate thickness of 3 to 5 micrometers, and stained with hematoxylin and eosin. Full histopathology was performed on the preserved organs and tissues of animals of Group 1 and 4 (Randomly selected five males and five females from each sex/ group). All gross lesions were examined microscopically. Microscopic examination of slides was done by Study Pathologist.

Postmortem examinations (offspring):

Dead pups were subjected to gross examination. Day 4 pups were sacrificed by thiopentone sodium and day 13 pups by CO2 asphyxiation and gross examination were performed.

Statistics:

The following statistical methods were used to analyze the body weight, feed consumption, hematological, biochemical parameters and organ weight data, Pre-coital interval, gestation length, litter size and litter weights, AGD, sex ratio and implantation sites, implantation losses, viability indices, offspring body weight and body weight change. Statistical analysis of pup body weight was based on individual pup data, taking litter size into account.
• Data was summarized in tabular form. Statistical analysis was performed using Stat Plus program
• The continuous data was checked for Normality with Shapiro-Wilk test and subjected to perform Analysis of Variance (ANOVA) to compare the difference between treated and control groups.
• Discontinuous data was subjected to non-parametric tests, for two groups Mann-Whitney U-Test and for more than two groups kruskal-wallis ANOVA was used.

Reproductive indices:

The following data was evaluated
 Mating performance and fertility (Pre-coital interval, mating index and pregnancy index)
 Gestation and parturition Data (Gestation length and parturition index)
 Litter Responses (Pre implantation loss, live/birth index and sex ratio)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male during treatment period significant increase in A/G and decrease in GLB was noticed in high dose (G4-1000 mg/kg body weight) group compared to control (G1-0 mg/kg body weight. In female significant increase Cholesterol in high dose (G4-1000 mg/kg body weight) was noticed compared to control group (G1-0 mg/kg body weight). The significance changes observed in biochemical parameters are non-dose dependent and no biological significant and hence could not be attributed to treatment related adverse effect.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The administration of Bisamid to Wistar rats by oral gavage at dose levels of 250, 500 and 1000 mg/kg body weight/day within the scope of OECD 422 revealed no treatment-related clinical signs, changes in the body weights or feed consumption. No treatment-related effects on reproduction/development such as mating index, fertility index, gestation length, post-natal loss, sex ratio and offspring growth and development. Macroscopic examination revealed no test item related findings in any of the animals of 250, 500 and 1000 mg/kg body weight body weight as group. No abnormality was observed attributable to test item in pups from all treatment groups. Microscopic examination revealed no abnormality attributable to test item - Bisamid at the highest dose tested i.e. 1000 mg/kg body weight. No Observed Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test: 1000 mg/kg body weight.

Executive summary:

The test item Bisamid, formulated in corn oil was administered by oral gavage to three groups, each of twelve male and twelve female Wistar rats, up to 42 days for males were treated, for two weeks premating phase, up to two weeks mating and post-mating. Females two weeks premating phase, up to two weeks mating, three weeks gestation and 13 days Iactation for females at dose levels of 250, 500 and 1000 mg/kg body weight/day. A control group of twelve males and twelve females were dosed with vehicle control (corn oil). The following observation and examinations were evaluated: Clinical signs (daily), body weights and feed consumption (once weekly except during mating). Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 13 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring body weights. Five male and five female animals selected for repeated dose toxicity screening. Males were sacrificed on day 43, followed by the sacrifice of all females on day 14 post-partum. Pups were sacrificed on Day 13 postpartum. Macroscopy at termination was performed for all animals. All males and females were evaluated for Hematology, Clinical biochemistry and urine analysis at the end of treatment period. Male and female animals were screened for T4 and TSH parameters from each dose group. Selected offspring were also screened for T4 and TSH parameters from each dose group. Testes, epididymides, seminal vesicle and prostate of all adult male; uterus, ovaries of all female animals and following organ weights were recorded on scheduled dates of necropsy from five male and five female animals selected for repeated dose toxicity screening. Wet weights were taken immediately after dissection to avoid drying. From all adult males on day 43 and females on day l4 postnatal and two pups from day 13 postnatal from each litter and thyroid weights was determined after fixation. All organ and tissue samples as defined under histopathology were processed, embedded and cut at an approximate to thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. Full histopathology was performed on the preserved argans and tissues of animals of group 1 and 4 (Randomly selected five males and five females from each sex/ group). All gross lesions were examined microscopically.

Results

Analytics:

Analysis of stability, homogeneity and dose concentration of the prepared dose formulations revealed acceptable levels. The dose formulation analytical report provided is appended to the study report.

Mortality:

There were no mortalities observed in control group (0 mg/kg body weight), low dose group (250 mg/kg body weight), intermediate dose group (500 mg/kg body weight) and high dose group (1000 mg/kg body weight) animals in premating, mating, gestation and lactation period. However, cannibalism was observed in one dam (Dam No. 021) of control dose group on day 2 lactation period. In low dose group (250 mg/kg body weight), cannibalism was observed in one dam on lactation day 1 in dam No, 037 and each one female in dam 42 and 48. Cannibalism was observed in high dose group (1000 mg/kg body weight) three female pups and one male on lactation day 3 in dam No, 091. The mortalities observed in Pups could not be attributed to test item which are common incidental findings in strain and species.

Clinical signs:

No clinical signs were observed in the control group (G1- 0 mg/kg body weight), low dose group (G2 - 250 mg/kg body weight), intermediate dose group (G3-500 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) in both male and female animals.

Detailed Clinical Examination and Functional observation battery:

No abnormalities were observed in all the animals in low (G2- 250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high dose group (G4-1000 mg/kg body weight) in both the sexes when compared with control animals. Functional observation battery parameters like grip strength (Forelimbs and hind limbs), Rotarod and IR Actimeter did not show any treatment related significant changes in all the treated groups when compared with control group.

Body weight:

Body weight and body weight gain of male and female of all treated animals were comparable with the control group animals.

Feed Consumption:

The significant changes were observed in feed consurnption in low dose (G2-250 mg/kg intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal and could not be attributed to test item administration.

Clinical Patbological Investigations:

The significance differences observed in hematology and clinical biochemistry parameters could not be attributed to treatment due to marginal increase or decrease in individual control values and no any biological significant. No treatment related significant differences were observed urinalysis parameter in all treated groups in both the sexes. No effects were detected on mating performance. All the female animals showed positive evidence mating. No significant difference was observed in the pre-coital interval of female animals between control and dose groups.

Reproductive indices:

Oestrous cyclicity was checked for all the female animals. Regular oestrous cycle was observed in all the animals of different groups in the study. Mating index for all the groups was 100% (as confirmed by presence of sperm in the vaginal smear). The two Dams from control (Gl-0 mg/kg body weight), two dams in low (G2-250 mg/kg body weight), intermediate dose (G3-500 mg/kg body weight) and one dam high dose (G4-1000 mg/kg body weight) was observed non pregnant. The pregnancy index for control (G 1- 0 mg/kg body weight), low (G2-250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high (G4 -1000 mg/kg body weight) was found to be 83.33, 83.33, 83.33 and 91.67, respectively. Of the litters delivered, in all the treatment groups, litter size at birth and on Day 1 and 4 post-partum were comparable to controls. No significant difference in the number of live births was noted between control and treated groups. There were no effects in fertility of treated animals.There were no differences in gestation lengths. The gestation length for treated females was comparable to controls. All animals showed gestation lengths between 21 to 24 days.The parturition index was 91.67 % in high dose group and whereas in control, low and intermediate dose it was observed 83.33 %. No significance changes were observed in post natal losses in the entire treated group in both sexes, sex ratio was calculated as percent (%) male. No significant difference in sex ratio was observed in any of the treated groups when compared with vehicle control. Offspring body weight on day 0, 4 and 13 were recorded. The significant changes were observed in Offspring body weight in intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal on day 0, 4 and 13 in male. The significant changes were observed in Offspring body weight in intermediate (G3-500 mg/kg body weight) are marginal on day 4 in female and could not be attributed to test item administration. No significant differences in the Anogenital distances (AGD) between control and treated male animals were observed. In females, the significant changes were observed in AGD in low (G2-250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal on day 4. In combined for both sexes, the significant changes were observed in AGD in low (G2-250 mg/kg body weight), intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) groups are marginal on day 4.

Terminal fasting body weights and organ weights:

Terminal fasting body weights, absolute and relative organ weights in males and females were comparable between control and high dose groups. Selected animals for organ collection: In males, absolute and relative organ weight of Kidneys and thymus belanging to intermediate (G3-500 mg/kg body weight) and high dose (G4-1000 mg/kg body weight) group was significantly decreased when compared with G 1. Absolute and relative organ weight of kidneys belonging to G2-250 mg/kg body weight) was significantly decreased as compared to G1. No significance was observed in terminal body weight, absolute and relative organ weight of randomly selected females when compared to control G1. Remaining animals: In males, absolute and relative organ weights of epididymides and prostate, seminal vesicle with coagulating glands belonging to G2, G3 and G4 was significantly decreased as compared to G1.Pups: Absolute and relative thyroid weight of pups belonging from G3 was significantly increased when compared with G1. No significance was observed in terminal body weight, absolute and relative organ weight of remaining females when compared to control G1. The all above significances can be considered as incidentalas there were no changes in gross and histopathology of the organs.

Macroscopig fndings

There were no treatment related macroscopic findings in males and females and in pups. Males: Two incidences of bilateral small sized testes in group 2 (Animal number 035 and 036) and one incidence in group 4 (Animal number 077) was observed. Microscopically slight to moderate degenerative changes in the seminiferous tubules of testes were observed in animal number 035 and 036. Marked seminiferous tubular atrophy was observed in animal number 077. Single incidence of bilateral small sized epididymides in group 4 (Animal number 077) showed marked oligospermia. One incidence of enlarged spleen in group 2 (Animal number 058) revealed extramedullary haematopoiesis on microscopic exarnination. Females: Single incidence of dilated uterus observed (Animal No.018) and was microscopically confirmed to be dilatation of the uterine lumen. This was considered to be a physiological change.

Microscopic findidings:

There were no treatment related microscopic findings in testes and epididymides in males and in ovaries in females at high dose. Males: In control group, two incidences of vacuolation of hepatocytes (Animal number 011 and 012) and single incidence of bile duct hyperplasia (Animal number 012) in liver was observed. Infiltration of mononuclear cells in kidneys (Animal number 012) was observed. Lungs revealed two incidences of increased alveolar macrophages, one in control (Animal number 009) and one in high dose group animal (Animal number 075), severity of lesion was comparable. Single incidence of infiltration of mononuclear cells was observed (Animal number 010). One incidence of vacuolation of zona fasciculata in control (Animal number 001) and one in high dose group animal (Animal number 084) was observed. Cell debri was found in the lumen of seminiferous tubules of testes of two control group animals (Animal number 011 and 012). One incidence of single cell necrosis and oligospermia in epididymides of control group animal (Animal number 010) was observed. High dose group animals revealed two incidences of infiltration of mononuclear cells in prostate gland (Animal number 079 and 084). Single incidence of cyst and pars distalis of pituitary gland was absoerved in control group animal (Animal number 010).Females: Two incidences of presence of alveolar macrophages in lung of control group animals was observed (Animal number 021 and 022). Single incidence of microscopic findings observed in various organs in control and treatment groups were considered incidental. These changes were not considered adverse as the incidences and severity was low and there were no changes in the weight of these organs. There were no treatment related changes in other vital organs such as liver, kidneys, heart, brain and reproductive organs such as testes and ovaries.

Conclusion:

The administration of Bisamid to Wistar rats by oral gavage at dose levels of 250, 500 and 1000 mg/kg body weight/day within the scope of OECD 422 revealed no treatment-related clinical signs, changes in the body weights or feed consumption. No treatment-related effects on reproduction/development such as mating index, fertility index, gestation length, post-natal loss, sex ratio and offspring growth and development. Macroscopic examination revealed no test item related findings in any of the animals of 250, 500 and 1000 mg/kg body weight body weight as group. No abnormality was observed attributable to test item in pups from all treatment groups. Microscopic examination revealed no abnormality attributable to test item - Bisamid at the highest dose tested i.e. 1000 mg/kg body weight. No Observed Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening Test: 1000 mg/kg body weight.