1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May 2022 to 31 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2023

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

EC Number:

257-913-4

EC Name:

1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Cas Number:

52434-90-9

Molecular formula:

C12H15Br6N3O3

IUPAC Name:

tris(2,3-dibromopropyl)-1,3,5-triazinane-2,4,6-trione

impurity 1

Reference substance name:

unknown impurities

IUPAC Name:

unknown impurities

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology Pvt. Ltd., Plot 4B, MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078.
- Females nulliparous and non-pregnant: Yes.
- Age at study initiation: 13 weeks at start of treatment.
- Weight at study initiation: Males body weight range 379.99 to 474.81 g; Females body weight range 228.69 to 297.87 g. At the commencement of the treatment, the weight variation of rats used did not exceed ± 20% of the mean body weight in each sex and group. For further details refer to the full study report.
- Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The fifth animal in the recovery groups of each sex was housed individually. During mating, two rats (one male and one female) were housed in standard polysulfone cages with a stainless-steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilized nesting material (paper shreds) was provided near-term (Gestation Day 20). Polycarbonate rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. These objects were provided during the pre-mating and post-mating period for males and during the pre-mating period for females and changed along with cage once a week. For recovery groups enrichment was provided throughout the experimental period.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before the pre-treatment period. During the acclimatization and pre-treatment period, animals were observed once daily for any abnormalities.

ENVIRONMENTAL CONDITIONS
Rats were housed in an environment-controlled room. The temperature maintained during the experiment ranged between 19.2 and 24.1°C, while relative humidity ranged between 64 and 67%. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.4–12.9 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 recorded from digital thermohygrometer.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Route of test item administration was through oral gavage. The oral route was chosen because it provides an exaggerated model of the normal exposure in humans.

Vehicle:

other: 0.5% Sodium carboxy methyl cellulose with 0.1% Tween 80 in Milli-Q water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Required quantities of the test item were weighed in a pre-calibrated beaker for each dose level separately and the vehicle (0.5% Sodium carboxy methyl cellulose with 0.1% Tween 80 in Milli-Q water) was added up to the pre-mark and mixed using a glass rod to get the final desired concentration of 10, 30 and 100 mg/mL for the G2, G3 and G4/G4R groups, respectively.
The dose formulations were prepared once daily before start of each day dosing or within the stability period. Homogeneity of the test item suspensions prior to sampling and during dosing was maintained by constant stirring using a magnetic stirrer.
The dose volume administered to each rat was an equivolume of 10 mL/kg bw throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were received from ‘Adgyl Lifesciences Private Limited’. For analysis, six replications (2 replicates from top, 2 from middle, and 2 from bottom layer) of each dose formulations of the G2, G3 and G4 groups and, in addition, duplicate samples of the vehicle control group (G1) were sampled. G2, G3 and G4 groups replicates were diluted in so as to obtain a final concentration of about 100 µg/mL of AP 729.
Similarly, the samples of vehicle control group (G1) were processed as per G2 group.
The samples were analysed for homogeneity and concentration of AP 729 on day 1 and on day 46 as per validated method.
Refer to full study report for all the details.

Duration of treatment / exposure:

Males were dosed for 49 days, up to and including the day before scheduled sacrifice (two weeks prior to mating, during mating period and approximately, two weeks post mating period).
Females were dosed throughout the treatment period for 50-62 days. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to lactation day 13).
Animals (males and females) in the recovery groups were dosed for 52 days and then kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period. These animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups (G1, G2, G3, G4): 10 males and 10 females
Recovery groups (G1R, G4R): 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 100 (G2), 300 (G3), 1000 (G4/G4R) mg/kg/day have been selected for this study based on the results of the 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No. N6519) and in consultation with the Sponsor. In addition to the test doses, a vehicle control (G1) and a vehicle control recovery group (G1R) were included.
- Rationale for animal assignment: Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM Software (Version 10.1.0.1, Instem LSS, Staffordshire ST15OSD, United Kingdom).
- Fasting period before blood sampling for clinical biochemistry: At the end of the pre-mating period, from randomly selected 5 males and 5 females of main groups (G1 to G4) and at the end of recovery period from all rats of recovery groups (G1R and G4R), which were fasted overnight (water allowed), approximately 3 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia.
- Rationale for selecting satellite groups: Animals in the recovery groups (G1R and G4R) were dosed for 52 days and then kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period. These animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity.
- Post-exposure recovery period in satellite groups: 14 days starting from the day of the first scheduled kill of dams.
- Dose range finding studies: Yes, 14-Day Repeated Dose Oral Toxicity Study in Wistar Rats (Study No. N6519).

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes. All rats were observed for morbidity and mortality twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays.

DETAILED CLINICAL OBSERVATIONS: Yes. Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period. All rats were observed inside the cage and outside the home cage in standard arena.

BODY WEIGHT: Yes. Individual body weights of males were recorded on Day 1 and at weekly (±1 Day) intervals thereafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males. All dams were weighed on gestation days 0, 7, 14 and 20 and on lactation days 0, 4 and 13.

FOOD CONSUMPTION: Food consumption (g) was measured at weekly intervals (±1 Day) during treatment and recovery period and was not measured during the cohabitation period. Food consumption of pregnant dams was measured on GD 0, 7, 14 and 20 and on Days 0, 4 and 13 of the lactation periods. Food consumption (g/rat/day) was calculated as the food consumed at each interval per cage divided by the number of rats per cage and the number of days in the interval period.

HAEMATOLOGY: Yes. At the end of the pre-mating period, from randomly selected 5 males and 5 females of main groups (G1 to G4) and at the end of recovery period from all rats of recovery groups (G1R and G4R), which were fasted overnight (water allowed), approximately 3 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia. The haematological parameters in Table 1 of section "Any other information on materials and methods incl. tables" were examined.

CLINICAL CHEMISTRY: Yes. At the end of the pre-mating period, from randomly selected 5 males and 5 females of main groups (G1 to G4) and at the end of recovery period from all rats of recovery groups (G1R and G4R), which were fasted overnight (water allowed), approximately 3 mL of blood was collected by retro-orbital plexus puncture method under isoflurane anaesthesia. The clinical chemistry parameters in Table 2 of section "Any other information on materials and methods incl. tables" were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes. Blood samples for hormone analysis were collected, and serum was separated, as per the following schedule for the determination of total T4 and TSH hormones:
• At least two available culled pups of either sex (if available) or of same sex per litter on LD 4.
• All dams prior to sacrifice and at least two available pups per litter on LD 13.
• All adult (parental) main groups males, prior to sacrifice at termination day.
In parental animals, approximately 1 mL of blood was collected without fasting by retro-orbital plexus puncture method under isoflurane anaesthesia for hormonal analysis. Pup blood was pooled by litter for thyroid hormone analyses. Pups were anesthetized with isoflurane and the jugular vein was incised at the neck region. The collected samples were pooled together for each litter.
Blood samples were collected in plain labelled tubes and kept on bench top till complete clot formation 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 labelled plastic tubes and stored at ~ -70 °C until analysed. The left-over samples from hormone analysis will be discarded within 3 months from sample collection.
Blood samples were not collected from the not littered females and from dams with all pups dead/cannibalized during lactation and sacrificed without blood collection.

URINALYSIS: Yes. 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 the recovery period from all rats of the recovery groups, in urine collection tubes. Each rat was placed overnight in a specially fabricated cage, fasted overnight (water allowed) and the next morning the collected urine was analysed for the parameters in Table 3 of section "Any other information on materials and methods incl. tables". Urine was also subjected to microscopic examination for sediments such as crystals, epithelial cells and casts.

NEUROBEHAVIOURAL EXAMINATION: Yes. Neurological examinations were performed on LD 13 for randomly selected 5 parental females of each main group, on day 46 for randomly selected 5 parental males of each main group and at the end of the recovery period (day 66) for all the recovery groups (G1R and G4R) animals. The following test and observations were performed: home cage observations, observations during removal of animal from home cage and handling, open field observations, functional tests (i.e. motor activity, sensory reactivity, landing hindlimbs foot splay, grip strength performance).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all adult animals were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. All the pre-terminally dead animals were subjected to detailed necropsy and findings were recorded.

HISTOPATHOLOGY: Yes, on completion of the gross pathology examination, the tissues/organs listed in Table 4 of section "Any other information on materials and methods incl. tables" were collected from all adults including recovery groups animals, and preserved in 10% neutral buffered formalin. In addition, the tissues/organs in Table 5 of section "Any other information on materials and methods incl. tables" were collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs), then weighed and preserved.
Histopathology examination was carried out on the preserved organs (including reproductive organs) from randomly selected 5 males and 5 females in the control and high dose groups and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. As there were no test item-related changes in the examined organs of high dose animals, tissues from remaining animals (lower dose group and recovery dose group animals) were not subjected for evaluation. Thyroids were examined from all adult males and females.

Statistics:

Data were captured using the Provantis(TM) laboratory information management system (LIMS). For further details refer to the full study report.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical sign of wound/abscess was observed in two female rats in the mid dose group. The wound/abscess was subjected to microscopic evaluation and was considered to be an incidental/spontaneous change. There were no treatment related clinical signs observed at any of the doses tested.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

An isolated incident of mortality was observed in a G3 group female just prior to necropsy on lactation Day 14. The animal was normal during morning observation. The cause of death could not be determined based on gross and microscopic evaluation.
There were no mortalities observed during the treatment at all the tested doses.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights were unaffected throughout treatment and recovery period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control groups. Moreover, treatment had no effects on the maternal body weights and body weight gains during gestation and lactation periods at all the doses when compared to the vehicle control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Significantly higher food consumption was observed during Days 29-36 at 1000 mg/kg/day in males and during Days 8-15 at 100 mg/kg/day and during Days 1-8 at 300 mg/kg/day in females. In the high dose recovery group, food consumption was significantly higher during Days 1-8 and 15-22 in males and during Days 1-8, 43-50 and 53-60 in females.
These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

Food efficiency:

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related alterations in any haematological parameters evaluated in male and female rats at all the doses.
Few significant differences (compared to vehicle control) noted among the tested groups were considered incidental as change was minimal in magnitude and lacked dose progression.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry evaluation in parental rats did not reveal any test item-related effects in any of the parameters in either sex.
All differences observed in biochemical parameters between control and test item treated rats, including the changes that attained statistical significance, were considered incidental in nature due to lack of dose progression and/or microscopic correlation.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes observed in thyroid stimulating hormone (TSH) and thyroxin hormone (T4) levels in adult males (at termination) and dams (on lactation day 13).

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes observed in any of the urine parameters analyzed in both males and females.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

During Functional Observation Battery (FOB), no treatment-related abnomarlities were observed in any of the doses tested in both sexes for home cage and handling observations, open field observations, sensory observations. The observed statistical variations in the motor activity measurements (i.e. resting time, ambulatory time and distance travelled) are considered to be incidental as there were no changes observed in the home cage or open field observations. Further there were no clinical signs observed during daily clinical observation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in terminal fasting body weights and organ weights at all dose levels tested in adult males and females.
A few variations in organ weights were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related gross findings in adult males and females.
All the observed gross findings were considered as incidental/spontaneous changes, not related to test item administration.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic changes in both adult males and females. Qualitative assessment of stages of spermatogenesis did not reveal any test item-related changes.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No treatment related effects on systemic, reproduction and fertility parameters.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

As there were no treatment related effects on systemic, reproduction, fertility parameters and F1 generation pups up to and including the highest dose tested 1000 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar rats for the test item AP 729 is determined to be 1000 mg/kg/day.

Executive summary:

The purpose of the present Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD Guideline 422) in Wistar rats administered by oral gavage was to determine the possible health hazards likely to arise from repeated exposure to AP 729 over a relatively limited period of time. Further, this study provides initial information on possible effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The present study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, following 14 days post treatment.

 

The test item was weighed and suspended in vehicle (0.5% Sodium carboxy methyl cellulose with 0.1% Tween 80 in Milli-Q water) and administered at the graduated dose levels of 100, 300 and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was an equivolume of 10 mL/kg. Each main group (G1, G2, G3, G4) in the experiment comprised 10 male and 10 female rats while each recovery group (G1R and G4R) comprised 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods for males, during pregnancy and up to Lactation Day (LD) 13 for females.

The homogeneity and stability of the test item in the vehicle were established before start of treatment at 1 and 100 mg/mL concentrations. Based on the results, the test item was stable in the vehicle up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on day 1 and during second month (day 46) of the treatment period. The results were considered acceptable, as the mean percent recovery was in the range 70% to 120% at each dose level and %RSD at each dose level was less than 20%.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period. After confirmation of mating by vaginal smear, the dams were weighed on presumed Gestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20. The littered dams were then weighed on LDs 0, 4 and 13 and the food consumption was recorded on LDs 0, 4 and 13.

The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4, after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13.

Neurological examinations (Functional Observation Battery tests, FOB) were conducted for randomly selected 5 females of each main group on LD 13, for randomly selected 5 males of each main group on treatment day 46 and towards the end of recovery period (day 66) for all the recovery group animals.

Laboratory investigations such as haematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and 5 parental females from each main group at the end of the two weeks pre-mating period, and towards the end of the recovery period for all the animals of the recovery groups, after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main groups males at termination, in all dams on LD 13 without fasting and from available pups on LD 4 and 13.

At sacrifice, the parental males (day 50), parental females (LD 14) and the recovery animals (day 67) were subjected to detailed necropsy after overnight fasting (water allowed) and tissues/organs were collected. The pups were sacrificed on LD 13 after examining the external genitals for signs of altered development.
Histopathology examination was carried out on the preserved organs (including reproductive organs) from randomly selected 5 males and 5 females in the control and high dose groups and on all gross lesions. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. As there were no test item-related changes in the examined organs of high dose animals, tissues from remaining animals (lower dose group and recovery dose group animals) were not subjected for evaluation. Thyroids were examined from all adult males and females and from one randomly selected male and one randomly selected female pup per litter on LD 13.

 

Under the adopted experimental conditions, the following results were obtained:

Clinical signs and Mortality: There were no treatment related clinical signs observed at any of the doses tested. There were no abnormalities observed in pups.
There were no mortalities observed during the treatment at all the tested doses. An isolated incident of mortality was observed in a G3 group female (Rab9323) just prior to necropsy on lactation day 14. The animal was normal during morning observation and the cause of death could not be determined based on gross/microscopic evaluation and was considered an incidental finding.

Functional Observation Battery (FOB): No treatment-related neurological abnormalities were observed at any of the doses tested.

Body weights: The mean body weights and body weight gains were unaffected by the treatment at all the tested doses in both sexes.

Food consumption: Treatment did not affect the food consumption at any of the tested doses in either sex.

Maternal body weights and food consumption: The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the tested doses.

Fertility parameters: Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.

Litter parameters: 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. No treatment-related changes in the ano-genital distance, ano-genital ratio, pup body weights were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on LD 13 at any of the doses tested.

Haematology, Coagulation, Clinical chemistry and Urine parameters: No test item-related changes were observed in the haematology, coagulation, clinical chemistry and urine parameters at all the doses tested in both sexes.

Hormone analysis: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item administration.

Terminal fasting body weights, organ weights and its ratios: There were no test item-related changes in terminal fasting body weights and organ weights at all dose levels tested in adult males and females. Pups body weights and lactation day 13 thyroid weights were unaffected by the treatment.

Gross and histopathology: There were no test item-related gross lesions observed in both the sexes. There were no developmental and non-developmental gross findings noted in male or female pups. Microscopically, there were no test item-related changes in both adult males and females. Qualitative assessment of stages of spermatogenesis did not reveal any test item-related changes. There were no test item-related microscopic changes in thyroid glands of pups. 
The cause of death of dead female rat (G3F-Rab9323) could not be determined based on gross and microscopic evaluation findings.

 

As there were no treatment related effects on systemic, reproduction, and fertility parameters and on F1 generation pups up to and including the highest dose tested of 1000 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar rats for the test item AP 729 is determined to be 1000 mg/kg/day under the adopted test conditions and doses tested.