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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

A 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 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.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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
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
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.
Route of administration:
oral: gavage
Vehicle:
other: 0.5% Sodium carboxy methyl cellulose with 0.1% Tween 80 in Milli-Q water
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1:1 ratio, male and female from the same dosing group.
- Length of cohabitation: Cohabitation was continued until there was evidence of sperms in the vaginal smear. All the females copulated successfully within ten days from the day of cohabitation.
- Proof of pregnancy: Sperm in vaginal smear, the day of confirmed mating was designated as gestation day (GD) 0.
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
Details on study schedule:
- Age at mating of the mated animals in the study: 15-16 weeks.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
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.
Parental animals: Observations and examinations:
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.

OTHER: Functional observation battery tests were performed on lactation day 13 for neurological parameters (i.e. subject’s response to handling and removal from home cage), open field observations, and functional tests (including motor activity, sensory evaluation, landing hindlimbs foot splay and measurement of grip performance).
Oestrous cyclicity (parental animals):
Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating 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.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.
Sperm parameters (parental animals):
Tissues/organs (including reproductive organs) collected from randomly selected 5 males in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis. All gross lesions were examined in all the groups.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes.
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.
On lactation day 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded. The number of pups born (litter size), sex and individual pup body weight of male and female pups were recorded on lactation days (LD) 0 and 4.
The ano-genital distance (AGD) of each pup was measured on LD 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
After standardization, the individual pup body weight was measured on LD 13 and the number of nipples/areolae in male pups was counted on LD 13.
All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
The litters were observed daily to note the number of alive, dead and cannibalized pups. In addition to daily clinical observations, all pups were observed for any abnormal behaviour.

GROSS EXAMINATION OF DEAD PUPS:
All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving parental male animals were sacrificed one day after the last treatment (day 50).
- Maternal animals: All surviving parental female animals were sacrificed one day after the last treatment (lactation day 14).

GROSS NECROPSY
All adult animals and pups 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. The number of implantation sites were recorded for all the dams.

HISTOPATHOLOGY/ORGAN WEIGHTS
On completion of the gross pathology examination, the tissues/organs listed in Table 1 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 2 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 and from one randomly selected male and one randomly selected female pup per litter on LD 13.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at lactation day 13.

GROSS NECROPSY
- Gross necropsy consisted of the examination of the external reproductive genitals for signs of altered development. Dead pups were examined for possible defects and/or cause of death and findings were recorded.
Statistics:
Data were captured using the Provantis(TM) laboratory information management system (LIMS). For further details refer to the full study report.
Reproductive indices:
The following indices were calculated:
- Male mating index (%)
- Male fertility index (%)
- Female mating index (%)
- Female fertility index (%)
- Mean number of implantations/group
- Post implantation loss (%)
Offspring viability indices:
The following indices were calculated:
- Mean litter size per group
- Mean viable litter size
- Live birth index (%)
- Day 4 survival index (%)
- Sex Ratio/ Percentage of male offspring (%)
- Ano-genital Distance Ratio (mm/g1/3)
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
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
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the mating and fertility indices of sires and dams at all the doses tested.
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.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no abnormalities observed in pups.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No treatment-related changes were observed in the survival rate of pups up to LD 4 at all the tested doses.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by the treatment at all the doses.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No changes attributable to the test item were detected in the ano-genital distance and ano-genital ratio in pups of either sex.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The male pups did not exhibit areola/nipple retention on PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Pups body weights and day 13 thyroid weights were unaffected by the treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Gross examination of pups did not reveal any abnormal findings.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic changes in thyroid glands of pups.
Other effects:
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 pups on lactation days 4 and 13 after birth.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No treatment related effects.
Key result
Reproductive effects observed:
no
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 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: 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.


 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Justification for classification or non-classification

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.

Additional information