N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

Key study: Test method OECD 422. GLP study. The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 4.3 mg/kg bw/day. The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in parental animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2020 – 22 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same 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:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 343.19-343.99 g (range of average values of each group of males at first day of dosing); 254.10-255.22 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 2 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 2 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-23.2ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was found insoluble in distilled water but formed uniform suspension with corn oil at the concentration of 1.9 mg/mL (considering highest dose of 9.5 mg/kg bw with a dose volume of 5 mL/kg bw) based on the in-house suspendibility test results. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 0.4, 0.86 and 1.9 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): L12017004, L32011001.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.4 mg/mL and 1.9 mg/mL in corn oil. Prepared test item formulations were administered to the animals within established stability conditions.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated HPLC method (study nº BIO-ANM 1513). Sampling and analysis of formulations were performed during week 1 (16-03-2020) and week 5 (14-04-2020) of the treatment. Samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 37 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.

Frequency of treatment:

Once a day

Details on study schedule:

F1 animals were not mated.

Dose / conc.:

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

Remarks:

G1 - Vehicle control + G1R - Vehicle Control Recovery Group

Dose / conc.:

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

Remarks:

G2 - Low dose

Dose / conc.:

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

Remarks:

G3 - Mid dose

Dose / conc.:

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

Remarks:

G4 - High dose + G4R - High dose Recovery Group

No. of animals per sex per dose:

Main Group: 12
Recovery Group: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses of 0, 50, 100 and 300 mg/kg bw /day were selected in a first DRF study (BIO-CTX 139) based on the available results for an acute oral toxicity study conducted as per OECD 423 guideline. Treatment related effects including mortalities were found at the lowest level tested. Thus, it was not possible to properly decide on doses for the main study and a second DRF study (BIO-CTX 171) was performed at doses of 0, 2.7, 8.3 and 25 mg/kg bw /day. The dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found and based on these results the doses 2, 4.3 and 9.5 mg/kg bw /day were considered for present main study.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations:
The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION:
yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice) and during the lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during the last week for the recovery group animals.
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals:
5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by a coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals:
5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine:
on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 37) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 65) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

OTHER:

Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 37 days of treatment).

Oestrous cyclicity (parental animals):

Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.

Sperm parameters (parental animals):

Parameters examined in all parental male in the control and high dose groups: testis and epididymis weight. Also, a detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 10 pups/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.
Also, blood samples were collected for measurement of serum T4 levels on the following schedule:
1. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
2. Two pups per litter (same sex) at termination (lactation day 13).

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after completion of 36 days of treatment.
- Maternal animals: All surviving animals on lactation day 14
- Recovery animals: All surviving animals after completion of 14 days observation from the first scheduled sacrifice of dams.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Tables 1 and 2 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed on lactation day 4 (those pups selected for blood collection) and on lactation day 13 (the rest of pups)
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- The dead pups and pups which were sacrificed on PND 4/13 were examined for gross abnormalities (external and internal examinations) with particular attention to the external reproductive genitals.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 4 below.

Reproductive indices:

See table 3 below.

Offspring viability indices:

See table 3 below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity observed at groups G1/G1R, G2 and G3 animals of either sex throughout the experimental period.

Animals in groups G4/G4R were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina. However, all the animals from group G4R started to recover from the above mentioned clinical signs during recovery period and found normal at termination. In group G4 all the animals did not reveal any clinical signs till day 23 of treatment period. However, from day 24 onwards the animals started to reveal clinical signs. In group G4R all the animals did not reveal any clinical signs till day 25 of treatment period. However, from day 26 onwards the animals started to reveal clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 at groups G2 and G3 in both sexes throughout the experimental period.

In group G4, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued til l termination. Even though these reductions were not statistically significant, these changes are considered as test item-related due to noted test item-related clinical signs and test item-related effects in neurological observations during these periods.

In group G4R, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till end of dosing period. Furthermore, some of these reductions were found to be statistically significant compared to the control group. These changes are considered as test item-related due to noted test item-related clinical signs and test item-related decreased mean feed consumption during these periods. However, the body weight and body weight gains were started to recover during recovery period in both sexes.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the gestation period which is statitistically significant on gestation day (GD) 14 and 20 for mean body weight and statitistically significant during GD 7 to 14 and 14 to 20 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the lactation period which is statitistically significant on lactation day (LD) 4, 7 and 13 for mean body weight and statitistically significant during LD 4 to 7 and 7 to 13 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption at groups G2 and G3 in both sexes during pre-mating, gestation and lactation periods when compared with vehicle control group.

In group G4, there were no changes noted in mean feed consumption in both sexes during pre-mating period when compared with vehicle control group. However, reductions in mean feed consumption were noted during gestation and lactation periods which are statitistically significant during GD 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13, when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item related reduction in body weights during these periods.

In group G4R, there were no changes noted in mean feed cosnumption till week 3 in both sexes. However, reduction in mean feed consumption was noted from week 4 onwards and continued till end of dosing period. These reductions are statistically significant during week 5 to 9 in G4R males; statistically significant during week 6 in G4R females when compared with respective vehicle control group. These changes are considered as test item-related due to noted test item-related clinical signs, decreased mean body weight and or percent body weight gain during these periods in both sexes. However, the mean feed consumption was started to recover during recovery period in both sexes.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals from all the tested dose and vehicle control main groups in both sexes during ophthalmological examination conducted towards end of the dosing period. Likewise, there were no ocular changes observed in any of the animals from both recovery groups in either sex during ophthalmological examination conducted towards end of the recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean haematology parameters in groups G2 and G3 of both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean absolute differential leucocyte count (nuetrophills, lymphocytes, monocytes, eosinophils, basophils), eosinophils (%), WBC count of group G4 males; decrease in mean RBC count, HGB, HCT, monocytes (absolute and %), prothrombin time of group G4 females; increase in mean reticulocyte count (absolute and %), eosinophils (absolute and %) and monocytes (%), decrease in mean lymphocytes (%) and prothrombin time of group G4R males; and decrease in RBC count and Activated Prothrombin Time, increase in MCV, MCH, reticulocyte count (absolute and %) of group G4R females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean clinical chemistry parameters of groups G2 and G3 in both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels of group G4 males; decrease in mean urea and blood urea nitrogen levels of group G4 females; decrease in mean creatinine levels and increase in mean calcium and phosphorous levels of group G4R males; increase in mean creatinine, sodium and total bilirubin levels and decrease in mean total cholesterol levels of group G4R females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in urinalysis parameters in any of the tested dose levels of both main and recovery groups when compared with vehicle control group. Some of the sporadically occurring statistically significant differences like, increase in mean urine pH levels and decrease in urine specific gravity levels in group G4R females were considered incidental and therefore of no toxicological relevance.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from groups G2 and G3 when compared with vehicle control groups.

In group G4, there were no changes noted in home cage/handling/open-field/sensory/ neuromuscular (hind limb foot splay)/physiological observations when compared with vehicle control group in both sexes. However, a slight (non-statistically significant) reduction in mean movements count during motor activity assessment in G4 males, statistically significant reduction in mean fore/hind limb grip strengths in G4 males were noted when compared with vehicle control group. These changes can be considered as secondary effects to the test item exposure, noted due to test item-related clinical signs, decreased mean body weight, percent body weight gain and mean feed consumption during this period.

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from group G4R in both sexes when compared with vehicle control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at group G4. The microscopic changes were characterized by mild decrease in lymphocytes in white pulp of spleen, cortical area of thymus and also mild decrease in marrow cellularity of femoral bone. Similar changes were not observed in lower and recovery group of animals. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). Few of the other microscopic findings observed in the study such as accessory cortical tissue in adrenal glands, ultimobranchial cysts(s) in thyroid gland and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of males and dams of main groups when compared with vehicle control group. The obtained T4 levels are within the normal range of this species and strain.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity of main group females during pre-mating period at any of the tested dose groups. The mean length of oestrus cycle (days) per dam during pre-mating period was unaffected at all the tested dose groups when compared with vehicle control group.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No effects were detected during examination of spermatogenesis stages in the male gonads and histopathology of interstitial testicular cell structure.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Gestation Index (%): A total of 11 (out of 11), 12 (out of 12), 11 (out of 11), and 9 (out of 11) females were confirmed with live born pups with a gestation index of 100.0%, 100.0%, 100.0% and 81.8% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group. However, in group G4 the noted reduction in gestation index is considered as test item-related, as 2 out of 11 fertile females were noted with total litter loss with evidence of implantation sites only and also this can be correlated with increased pup mortalities and reduced pup survival index from other littered females of same dose group.

Post-Implantation Loss (%): A mean number of 0.18, 0.25, 0.18 and 5.11 post-implantation losses with a percentage of 1.82%, 2.68%, 1.53% and 38.27% were noted from groups G1, G2, G3 and G4, respectively. In group G4, the post-implantation loss was statistically significant in both number and percentage when compared with vehicle control group. This noted change is considered as test item-related, due to noted increase in number of resorptions, test item-related effects in litter observations or test item-related maternal effects during gestation and lactation periods.

Post-Natal Loss (%): In group G4, the mean number of post-natal loss was noted as 4.67 per litter (0 in control group) with a percentage of 62.47% (0% in control group). These noted increase in post-natal losses were statistically significant in both number and percentage when compared with vehicle control group. These effects are considered as test item-related, due to noted test item-related increase in number of pup mortalities, increased resorptions, effects in litter observations during lactation period, reduced pup survival index, reduced pup weights and also due to noted test item-related maternal effects during gestation and lactation periods.

The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Remarks on result:

other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

immune system

Organ:

bone marrow

spleen

thymus

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no external anomalies or no behaviour changes noted in any of the pups during daily observation of pups from tested dose groups G2, G3 and vehicle control group litters during post-natal period. All the pups were noted with normal behaviour during daily observations.

In group G4, test item-related external anomalies and mortalities were noted during post-natal period. These changes are considered as test item-related due to noted effects on maternal body weights and feed consumption of litters at this dose level and also these findings can be correlate with reduced mean pup weights.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In group G4, reduced litter size at birth, reduced live birth index per litter, reduced number of live pups during lactation period in either sex, increased number of dead pups at birth and during lactation period and reduced pup survival index during lactation period were noted when compared with vehicle control group. These noted changes are statistically significant for number of live born female pups, number of survived male pups per litter during LD 1 to 4, number of survived female pups per litter during LD 1 to 4, 5 to 7 and 8 to 13, number of dead pups per litter during LD 1 to 4 and 5 to 7 and pup survival index between LD 1 to 4, 5 to 7 and 8 to 13 when compared with vehicle control group. These noted changes at group G4 are considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group G4, statistically significant reduction in mean pup (both male and female) weight per litter on PND 1, 4, 7 and 13 were noted. These changes are considered as test item-related due to increased pup mortalities, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

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):

effects observed, treatment-related

Description (incidence and severity):

In group G4, reduction in mean pup (both male and female) anogenital distance measurement (mm) and ratio per litter were noted. Changes in AGD ratio were found statistically significant in both sexes. These changes are considered as test item-related due to increased pup mortalities, decreased growth/weight of pups, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences of nipple/arealaes in any of the male pups examined on their PND 13 from any of the tested dose group litters and vehicle control group litters.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, the dead pups which were found dead by birth and during lactation period noted with pale discoloration and red discoloration at nasal region from group G4 litters. No gross pathological changes were noted in any of the survived pups from group G4 and all pups from other dose group litters.

Histopathological findings:

not examined

Description (incidence and severity):

The histopathological examination of the thyroid gland pups was not conducted as there were no changes noted in this organ during miscoscopic examination and no effects noted in absolute or relative weight in adults of both sexes.

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of PND 13 pups in any of the tested dose groups when compared with vehicle control group. The obtained T4 levels of PND 13 pups are within the normal range of this species and strain. There were no changes noted in T4 levels of PND 4 pups from the tested dose groups G2 and G3 when compared with vehicle control group. In group G4, serum T4 levels of PND 4 pups was not conducted as there were no surplus pups remained from this dose level on PND 4. However, the obtained T4 levels of PND 4 pups from groups G2 and G3 are within the normal range of this species and strain.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in parental animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

Remarks on result:

other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 5. Summary of vaginal smear examination for determination of oestrus cyclicity

Determination of Oestrus Cyclicity during Pre-mating Treatment Period
Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		Females with Complete Regular Cycles	Females with at least one Irregular Oestrus Cycle	Average Length of Oestrous Cycle (Days)
G1, F & 0	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G2, F & 2	12	n	12	0	Mean	4.92
		%	100.00	0.00	±SD	0.19
					n	12
G3, F & 4.3	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G4, F & 9.5	12	n	12	0	Mean	5.00
		%	100.00	0.00	±SD	0.00
					n	12

Table 6. Summary record of reproductive performance

Group, Sex & Dose   (mg/kg body weight/day)	No. of Males with Evidence of Mating (No. of Males used for Mating)	Male Mating Index (%)	No. of Males Capable of Impregnating a Female (No. of males used for Mating)	Male Fertiltiy Index (%)
G1, M & 0	11 (12)	91.7	11 (12)	91.7
G2, M & 2	11 (12)	91.7	11 (12)	91.7
G3, M & 4.3	11 (12)	91.7	11 (12)	91.7
G4, M & 9.5	11 (12)	91.7	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)		Cohabitation Record and Pre-coital Interval (Mean Time to Mating)				Gestational Length (duration of pregnancy)
		Pre-coital Interval (Days)		Concieving Days (1 to 5)	Concieving Days (More than 5 days)	Gestation Length (Days)
G1, F & 0	Mean	9.08	n	5	7	22.09
	±SD	6.05	%	41.67	58.33	0.54
	n	12				11
G2, F & 2	Mean	6.75	n	7	5	22.17
	±SD	5.33	%	58.33	41.67	0.39
	n	12				12
G3, F & 4.3	Mean	7.58	n	6	6	22.18
	±SD	5.70	%	54.55	54.55	0.60
	n	12				11
G4, F & 9.5	Mean	6.33	n	7	5	22.11
	±SD	5.38	%	58.33	41.67	0.33
	n	12				9

n: Number of dams

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)		Female Fertiltiy Index (%)
	No. of Females with Evidence of Mating (No. of Females used for Mating)	Female Mating Index (%)	No. of Females Confirmed as Fertile (No. of Females used for Mating)	Female Fertiltiy Index (%)
G1, F & 0	12 (12)	100.0	11 (12)	91.7
G2, F & 2	12 (12)	100.0	12 (12)	100.0
G3, F & 4.3	12 (12)	100.0	11 (12)	91.7
G4, F & 9.5	12 (12)	100.0	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)	Female Fecundity or Pregnancy Index (%)			Gestation Index (%)
	No. of Pregnant Females	No. of Females Confirmed with Mating	Female Fecundity or Pregnancy Index (%)	Females with Live Born Pups at Parturation	No. of Females with Evidence of Pregnancy	Gestation Index (%)
G1, F & 0	11	12	91.7	11	11	100.0
G2, F & 2	12	12	100.0	12	12	100.0
G3, F & 4.3	11	12	91.7	11	11	100.0
G4, F & 9.5	11	12	91.7	9	11	81.8

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)				Post-natal Loss (%)
		No. of Implantations	No. of Viable Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)	Total No. of Deaths/ Cannibalized during Lactation Period	Post-natal Loss (%)
G1, F & 0	Mean	10.82	10.64	0.18	1.82	0.00	0.00
	±SD	1.54	1.69	0.40	4.05	0.00	0.00
	n	11	11	11	11	11	11
G2, F & 2	Mean	10.67	10.42	0.25	2.68	0.00	0.00
	±SD	1.78	1.98	0.62	7.32	0.00	0.00
	n	12	12	12	12	12	12
G3, F & 4.3	Mean	11.00	10.82	0.18	1.53	0.00	0.00
	±SD	1.67	1.60	0.40	3.41	0.00	0.00
	n	11	11	11	11	11	11
G4, F & 9.5	Mean	12.56	7.44	5.11*	38.27*	4.67*	62.47*
	±SD	2.07	3.13	4.37	28.23	4.30	44.96
	n	9	9	9	9	9	9

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7. Summary of delivery and litter observation record during lactation period

Group, Sex & Dose (mg/kg body weight/day)		Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex Ratio (m/f) at Birth	Live Birth Index (%)
			Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total
G1, F & 0	Mean	10.73	5.55	5.09	10.64	0.09	0.00	0.09	0.00	0.00	0.00	0.00	1.34	99.09
	±SD	1.62	2.11	1.64	1.69	0.30	0.00	0.30	0.00	0.00	0.00	0.00	1.01	3.02
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	10.50	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.08	0.00	0.08	1.15	99.40
	±SD	2.11	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.29	0.00	0.29	1.28	2.06
	n	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	10.91	5.09	5.73	10.82	0.09	0.00	0.09	0.00	0.00	0.00	0.00	0.99	99.30
	±SD	1.70	1.30	1.68	1.60	0.30	0.00	0.30	0.00	0.00	0.00	0.00	0.44	2.32
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	8.11	4.00	3.44*	7.44	0.33	0.33	0.67	0.00	0.00	0.00	0.00	1.17	90.43
	±SD	2.67	1.73	2.24	3.13	0.71	0.71	1.32	0.00	0.00	0.00	0.00	0.86	21.83
	n	9	9	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

*: Statistically significant (P<0.05) change than the vehicle control group

Note: A total of 9 dams were considered for mean calculations of all end points/parameters at birth from group G4 as 1 out of 12 dams confirmed as non-pregnant; 2 out of 12 dams were confirmed with total litter loss [postimplantation loss].

Group, Sex & Dose (mg/kg body weight/day)		During LD 1 to 4									Sex Ratio (m/f) on LD 4	Pup Survival Index (%) LD 1 to 4
		Live Pups (No.)			Dead Pups (No.)			Cannibalized (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	5.55	5.09	10.64	0.00	0.00	0.00	0.00	0.00	0.00	1.34	100.00
	±SD	2.11	1.64	1.69	0.00	0.00	0.00	0.00	0.00	0.00	1.01	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.00	0.00	1.15	100.00
	±SD	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.00	0.00	1.28	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	5.73	10.82	0.00	0.00	0.00	0.00	0.00	0.00	0.99	100.00
	±SD	1.30	1.68	1.60	0.00	0.00	0.00	0.00	0.00	0.00	0.44	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	1.89*	1.22*	3.11*	2.11*	2.11*	4.22*	0.00	0.11	0.11	0.85	40.89*
	±SD	2.42	1.39	3.72	2.47	2.37	4.29	0.00	0.33	0.33	1.13	46.69
	n	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

*: Statistically significant (P<0.05) change than the vehicle control group;

Note: A total of 9 dams were considered for mean calculations of all end points/parameters during LD 1 to 4 from group G4 as 1 out of 12 dams confirmed as non-pregnant;

2 out of 12 dams were confirmed with total litter loss [postimplantation loss].

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	Pups Sacrificed for Blood Collection on LD 4 (No.)			Live Pups (No.) on LD 4 after Sacrificed for Blood Collection			During Lactation Day 5 to 7									Sex Ratio (m/f) on LD 7	Pup Survival Index (%) during LD 5 to 7
								Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibilized Pups per litter (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Female
G1, F & 0	Mean	0.00	0.55	0.55	5.55	4.55	10.09	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	0.00	0.82	0.82	2.11	1.44	1.04	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	0.00	0.75	0.75	4.58	5.08	9.67	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.00	0.97	0.97	0.90	1.62	1.30	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	0.00	1.00	1.00	5.09	4.73	9.82	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	0.00	0.89	0.89	1.30	1.42	0.98	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	0.00	0.00	0.00	3.40	2.20	5.60	3.40	1.80*	5.20	0.00	0.40*	0.40*	0.00	0.00	0.00	1.70	77.50*
	±SD	0.00	0.00	0.00	2.30	1.10	3.21	2.30	1.30	3.42	0.00	0.55	0.55	0.00	0.00	0.00	1.20	43.66
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

n: Number of Dams; LD: Lactation Day; m/f: Male/Female

*: Statistically significant (P<0.05) change than the vehicle control group

Note: A total of 5 dams were considered for mean calculations of all end points/parameters during LD 5 to 7 from group G4 as 4 out of 9 dams were noted with total postnatal pup loss during LD 1 to 4.

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	During Lactation Day 8 to 13									Sex Ratio (m/f) at LD 13	Pup Survival Index (%) during LD 8 to 13
		Live Pups (No.)			Dead Pups (No.)			Cannibalized (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	4.25	2.00*	6.25	0.00	0.25	0.25	0.00	0.00	0.00	2.38	97.22*
	±SD	1.50	0.82	1.71	0.00	0.50	0.50	0.00	0.00	0.00	0.95	5.56
	n	4	4	4	4	4	4	4	4	4	4	4

n: Number of Dams; LD: Lactation Day; m/f: Male/Female;

*: Statistically significant (P<0.05) change than the vehicle control group

Note: A total of 4 dams were considered for mean calculations of all end points/parameters during LD 8 to 13 from group G4 as 1 out of 5 dams were noted with total postnatal pup loss during LD 5 to 7.

Table 8. Summary record of pup observations during post-natal period

Group, Sex & Dose (mg/kg body weight/day)		At Birth (PND 1)	PND 1 to 4	Pups Sacrificed for Blood Collection on PND 4 (No.)*	PND 5 to 7^$	PND 8 to 13^$
G1, F & 0	No. of Dams^#	11	11	4	11	11
	No. of Live Pups	117	117	6	111	111
	Pup Observation/ No. of Pups observed	N/117	N/117	-	N/111	N/111
G2, F & 2	No. of Dams^#	12	12	5	12	12
	No. of Live Pups	125	125	9	116	116
	Pup Observation/ No. of Pups observed	N/125	N/125	-	N/116	N/116
G3, F & 4.3	No. of Dams^#	11	11	7	11	11
	No. of Live Pups	119	119	11	108	108
	Pup Observation/ No. of Pups observed	N/119	N/119	-	N/108	N/108
G4, F & 9.5	No. of Dams^#	9	9	0	9	9
	No. of Live Pups	67	28	0	26	25
	Pup Observation/ No. of Pups observed	P/28; HS/1; N/38	L/2; N/26	-	L, P/1; N/25	N/25

N: Normal; PND: Post-natal Day; #: Dams confirmed with littering; P: Pale colored; HS: Haemorrhagic spot; L: Lethargic

*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded

Table 9. Summary record of mean pup weight (g) per litter

Group, Sex & Dose (mg/kg body weight/day)		PND 1		PND 4		PND 7		PND 13
		Mean Pup Weight (g)		Mean Pup Weight (g)		Mean Pup Weight (g)		Mean Pup Weight (g)
		Male	Female	Male	Female	Male	Female	Male	Female
G1, F & 0	Mean	6.75	6.44	11.42	10.64	16.25	15.26	29.26	27.87
	±SD	0.09	0.17	0.36	0.20	0.47	0.35	0.51	0.82
	n	11	11	11	11	11	11	11	11
G2, F & 2	Mean	6.82	6.57	11.58	10.81	16.51	15.60	29.58	28.56
	±SD	0.14	0.16	0.37	0.19	0.37	0.40	0.25	0.40
	n	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	6.78	6.48	11.50	10.72	16.54	15.44	29.47	28.13
	±SD	0.12	0.18	0.32	0.23	0.30	0.33	0.33	0.65
	n	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	3.94*	3.73*	6.95*	5.70*	10.88*	9.11*	19.99*	17.88*
	±SD	0.89	0.80	1.79	1.91	3.28	2.75	4.47	4.49
	n	9	8	4	5	4	4	4	4

n: Number of litters

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10. Summary record of mean pup ano-genital distance (AGD) measurement (mm) and ano-genital distance (AGD) ratio per litter on post-natal day 4

Group, Sex & Dose (mg/kg body weight/day)		Mean Male Pup AGD Measurement (mm)	Mean Female Pup AGD Measurement (mm)	Mean Male Pup AGD Ratio	Mean Female Pup AGD Ratio
G1, F & 0	Mean	5.43	2.69	2.41	1.22
	±SD	0.22	0.12	0.08	0.05
	n	11	11	11	11
G2, F & 2	Mean	5.42	2.77	2.39	1.25
	±SD	0.25	0.13	0.09	0.05
	n	12	12	12	12
G3, F & 4.3	Mean	5.34	2.70	2.37	1.23
	±SD	0.19	0.13	0.07	0.05
	n	11	11	11	11
G4, F & 9.5	Mean	4.07	1.78	2.14*	1.00*
	±SD	0.61	0.41	0.15	0.15
	n	4	5	4	5

n: Number of litters

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group, Sex & Dose (mg/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Post-natal Day 13
G1, F & 0	Mean	0.00
	±SD	0.00
	n	11
G2, F & 2	Mean	0.00
	±SD	0.00
	n	12
G3, F & 4.3	Mean	0.00
	±SD	0.00
	n	11
G4, F & 9.5	Mean	0.00
	±SD	0.00
	n	9

n: Number of litters

Conclusions:

The NOAEL of the test substance for parental and reproductive toxicity in rats by oral route was determined to be 4.3 mg/kg bw/day. The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in parental animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance POLY-U by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 2, 4.3 and 9.5 mg/kg bw/d based on the results of two consecutive dose range finding studies performed with the same species for a period of 14 days. at doses of 50, 100 and 300 mg/kg bw /d and 2.7, 8.3 and 25 mg/kg bw /d respectively. The test item was administered to the main group males for a period of 37 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and for PND 4 (from available pups) and PND 13 pups.

In groups G2 and G3, there were no indication of test item-related effects in any of the parameters assessed for systemic or reproductive toxicity.

In group G4/G4R, the animals were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina, as well as test item-related reduction in body weight and feed consumption. Also, test item-related reduced motor activity assessments, effects on clinical pathology investigations and reduced thymus spleen and liver weights were found at the dose level of 9.5 mg/kg bw/d. During gross pathology, test item-related changes like wet pernineum (4/12 males and 7/12 females), wet pernineum along with blood stains around vagina (2/12 females) and uterus with resorptions/dead fetuses (2/12 females) were noted from group G4. Microscopically, test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at this dose level. Thus, the investigations were extended to the lower dose and recovery groups for these organs. Effects were not observed in these groups. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). The high dose recovery group animals treated with 9.5 mg/kg bw/day were recovered from the systemic toxicity effects during recovery period.

In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. Also, test item-related external anomalies in pups, test item-related reduced mean pup weight and ano-genital distance ratio per litter in either sex were noted. However, there were no occurrences of nipples in male pups examined on PND 13 from all the litters. The pups which were found dead by birth and during postnatal period were noted with gross pathological changes like pale coloured and nose discoloration. No gross pathological changes were noted in any of the survived pups. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 pups.

However, these reproductive effects were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Therefore, based on the above results discussed, the NOAEL of the test item POLY-U for parental and reproductive toxicity is considered to be 4.3 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

4.3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A key study is available with a Klimisch score of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Key study: Test method OECD 422. GLP study. The NOAEL of the test substance for maternal and developmental toxicity in rats by oral route was determined to be 4.3 mg/kg bw/day. The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in maternal animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 February 2020 – 22 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: 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:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 343.19-343.99 g (range of average values of each group of males at first day of dosing); 254.10-255.22 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 2 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 2 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6-23.2ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was found insoluble in distilled water but formed uniform suspension with corn oil at the concentration of 1.9 mg/mL (considering highest dose of 9.5 mg/kg bw with a dose volume of 5 mL/kg bw) based on the in-house suspendibility test results. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 0.4, 0.86 and 1.9 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): L12017004, L32011001.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.4 mg/mL and 1.9 mg/mL in corn oil. Prepared test item formulations were administered to the animals within established stability conditions.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated HPLC method (study nº BIO-ANM 1513). Sampling and analysis of formulations were performed during week 1 (16-03-2020) and week 5 (14-04-2020) of the treatment. Samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 37 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation.

Frequency of treatment:

Once a day

Duration of test:

Main group Males: 17 March - 23 April 2020
Main group Females: 17 March - 23 May 2020
Recovery Animals: 17 March - 21 May 2020

Dose / conc.:

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

Remarks:

G1 - Vehicle control + G1R - Vehicle Control Recovery Group

Dose / conc.:

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

Remarks:

G2 - Low dose

Dose / conc.:

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

Remarks:

G3 - Mid dose

Dose / conc.:

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

Remarks:

G4 - High dose + G4R - High dose Recovery Group

No. of animals per sex per dose:

Main Group: 12
Recovery Group: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Doses of 0, 50, 100 and 300 mg/kg bw /day were selected in a first DRF study (BIO-CTX 139) based on the available results for an acute oral toxicity study conducted as per OECD 423 guideline. Treatment related effects including mortalities were found at the lowest level tested. Thus, it was not possible to properly decide on doses for the main study and a second DRF study (BIO-CTX 171) was performed at doses of 0, 2.7, 8.3 and 25 mg/kg bw /day. The dose of 25 mg/kg bw /d was the lowest dose level at which treatment related adverse effects were found and based on these results the doses 2, 4.3 and 9.5 mg/kg bw /day were considered for present main study.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (water allowed).

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations:
The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION:
yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, at the end of the dosing period for males (shortly prior to scheduled sacrifice) and during the lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during the last week for the recovery group animals.
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals:
5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by a coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
on the day of scheduled terminal sacrifice (for main group males on day 38, for main group females on lactation day 14 and for recovery group animals on the day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals:
5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Na/K/Cl analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine:
on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 37) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 65) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice:
Male animals: All surviving animals after completion of 36 days of treatment.
Maternal animals: All surviving animals on lactation day 14
Recovery animals: All surviving animals after completion of 14 days observation from the first scheduled sacrifice of dams.
- Organs examined: See tables 1 and 2 below.

OTHER:
Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 37 days of treatment).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes.
- Number of implantations: Yes.
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Ovaries and uterus were examined for histopathology.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: No data
- Head examinations: No data

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 4 below.

Indices:

See table 3 below.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity observed at groups G1/G1R, G2 and G3 animals of either sex throughout the experimental period.

Animals in groups G4/G4R were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina. However, all the animals from group G4R started to recover from the above mentioned clinical signs during recovery period and found normal at termination. In group G4 all the animals did not reveal any clinical signs till day 23 of treatment period. However, from day 24 onwards the animals started to reveal clinical signs. In group G4R all the animals did not reveal any clinical signs till day 25 of treatment period. However, from day 26 onwards the animals started to reveal clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 at groups G2 and G3 in both sexes throughout the experimental period.

In group G4, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued til l termination. Even though these reductions were not statistically significant, these changes are considered as test item-related due to noted test item-related clinical signs and test item-related effects in neurological observations during these periods.

In group G4R, there were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 till week 3 in both sexes. However, reduction in mean body weight and percent change in mean body weight gain was noted from week 4 onwards and continued till end of dosing period. Furthermore, some of these reductions were found to be statistically significant compared to the control group. These changes are considered as test item-related due to noted test item-related clinical signs and test item-related decreased mean feed consumption during these periods. However, the body weight and body weight gains were started to recover during recovery period in both sexes.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the gestation period which is statitistically significant on gestation day (GD) 14 and 20 for mean body weight and statitistically significant during GD 7 to 14 and 14 to 20 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

In group G4, reduction in mean body weight and percent change in mean body weight gain was noted throughout the lactation period which is statitistically significant on lactation day (LD) 4, 7 and 13 for mean body weight and statitistically significant during LD 4 to 7 and 7 to 13 for percent change in mean body weight gain when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item-related reduction in feed consumption during these periods.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption at groups G2 and G3 in both sexes during pre-mating, gestation and lactation periods when compared with vehicle control group.

In group G4, there were no changes noted in mean feed consumption in both sexes during pre-mating period when compared with vehicle control group. However, reductions in mean feed consumption were noted during gestation and lactation periods which are statitistically significant during GD 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13, when compared with vehicle control group. These reductions are considered as test item-related due to noted test item-related clinical signs and test item related reduction in body weights during these periods.

In group G4R, there were no changes noted in mean feed cosnumption till week 3 in both sexes. However, reduction in mean feed consumption was noted from week 4 onwards and continued till end of dosing period. These reductions are statistically significant during week 5 to 9 in G4R males; statistically significant during week 6 in G4R females when compared with respective vehicle control group. These changes are considered as test item-related due to noted test item-related clinical signs, decreased mean body weight and or percent body weight gain during these periods in both sexes. However, the mean feed consumption was started to recover during recovery period in both sexes.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals from all the tested dose and vehicle control main groups in both sexes during ophthalmological examination conducted towards end of the dosing period. Likewise, there were no ocular changes observed in any of the animals from both recovery groups in either sex during ophthalmological examination conducted towards end of the recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in mean haematology parameters in groups G2 and G3 of both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean absolute differential leucocyte count (nuetrophills, lymphocytes, monocytes, eosinophils, basophils), eosinophils (%), WBC count of group G4 males; decrease in mean RBC count, HGB, HCT, monocytes (absolute and %), prothrombin time of group G4 females; increase in mean reticulocyte count (absolute and %), eosinophils (absolute and %) and monocytes (%), decrease in mean lymphocytes (%) and prothrombin time of group G4R males; and decrease in RBC count and Activated Prothrombin Time, increase in MCV, MCH, reticulocyte count (absolute and %) of group G4R females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean clinical chemistry parameters of groups G2 and G3 in both sexes when compared with vehicle control group. In group G4/G4R, the following statistically significant changes were noted: decrease in mean alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels of group G4 males; decrease in mean urea and blood urea nitrogen levels of group G4 females; decrease in mean creatinine levels and increase in mean calcium and phosphorous levels of group G4R males; increase in mean creatinine, sodium and total bilirubin levels and decrease in mean total cholesterol levels of group G4R females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes observed in urinalysis parameters in any of the tested dose levels of both main and recovery groups when compared with vehicle control group. Some of the sporadically occurring statistically significant differences like, increase in mean urine pH levels and decrease in urine specific gravity levels in group G4R females were considered incidental and therefore of no toxicological relevance.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from groups G2 and G3 when compared with vehicle control groups.

In group G4, there were no changes noted in home cage/handling/open-field/sensory/ neuromuscular (hind limb foot splay)/physiological observations when compared with vehicle control group in both sexes. However, a slight (non-statistically significant) reduction in mean movements count during motor activity assessment in G4 males, statistically significant reduction in mean fore/hind limb grip strengths in G4 males were noted when compared with vehicle control group. These changes can be considered as secondary effects to the test item exposure, noted due to test item-related clinical signs, decreased mean body weight, percent body weight gain and mean feed consumption during this period.

There were no changes noted in neurological/functional examinations like home cage/ handling/open-field/sensory/neuromuscular (hind limb foot splay)/physiological observations and grip strength or motor activity assessments in any of the animals from group G4R in both sexes when compared with vehicle control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There were no changes noted in both mean absolute and relative organ weights of groups G2 and G3 in either sex when compared with vehicle control group.

In group G4, statistically significant decrease in mean thymus weight (absolute and relative) of males, statistically significant decrease in mean spleen weight (absolute), mean liver weight (absolute) and thymus weight (absolute and relative) of females was noted when compared with vehicle control group. These observed changes can be considered as test item-related due to noted mild decrease in lymphocytes in white pulp of spleen and cortical area of thymus during microscopic examination of these organs at this dose level. These changes can also correlate with test item-related and statistically significant reduction in mean terminal body weight at this dose level.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no gross pathological changes observed in any of the adult animals from groups G1/G1R, G2 and G3.

The animals of group G4 were noted with test item related external gross pathological changes like wet pernineum (4 males and 7 females), wet pernineum along with blood stains around vagina (2 females) and test item related internal gross pathological changes like uterus with resorptions/dead fetuses during necropsy. However, the group G4R animals of both sexes treated with high dose did not reveal any external or internal gross pathological changes during necropsy. These observations are because of recovery of animals from clinical signs during recovery period and hence all the animals were found normal at termination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at group G4. The microscopic changes were characterized by mild decrease in lymphocytes in white pulp of spleen, cortical area of thymus and also mild decrease in marrow cellularity of femoral bone. Similar changes were not observed in lower and recovery group of animals. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). Few of the other microscopic findings observed in the study such as accessory cortical tissue in adrenal glands, ultimobranchial cysts(s) in thyroid gland and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of males and dams of main groups when compared with vehicle control group. The obtained T4 levels are within the normal range of this species and strain.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions ocurred in any dose group.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

A mean number of 0.18, 0.25, 0.18 and 5.11 post-implantation losses with a percentage of 1.82%, 2.68%, 1.53% and 38.27% were noted from groups G1, G2, G3 and G4, respectively. In group G4, the post-implantation loss was statistically significant in both number and percentage when compared with vehicle control group. This noted change is considered as test item-related, due to noted increase in number of resorptions, test item-related effects in litter observations or test item-related maternal effects during gestation and lactation periods.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

In Group 4, 1 out of 11 fertile females were confirmed with total litter loss with evidence of resorptions at necropsy.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

In Group 4, 1 out of 11 fertile females were confirmed with early/late resorptions.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

In Group 4, 1 out of 11 fertile females were confirmed with total litter loss with evidence of dead fetuses at necropsy.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean gestation length (day of confirmed as mated to day of parturition) was 22.09, 22.17, 22.18 and 22.11 days for groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the control group.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

A total of 11 (out of 12), 12 (out of 12), 11 (out of 12), and 11 (out of 12), females were confirmed as pregnants / with evidence of implantation sites with a fecundity index of 91.7%, 100.0%, 91.7% and 91.7% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the control group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Oestrous cycle: There were no irregularities observed in the oestrus cyclicity of main group females during pre-mating period at any of the tested dose groups. The mean length of oestrus cycle (days) per dam during premating period was unaffected at all the tested dose groups when compared with control group.

Gestation Index (%): A total of 11 (out of 11), 12 (out of 12), 11 (out of 11), and 9 (out of 11) females were confirmed with live born pups with a gestation index of 100.0%, 100.0%, 100.0% and 81.8% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group. However, in group G4 the noted reduction in gestation index is considered as test item-related, as 2 out of 11 fertile females were noted with total litter loss with evidence of implantation sites only and also this can be correlated with increased pup mortalities and reduced pup survival index from other littered females of same dose group.

Details on maternal toxic effects:

The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

pre and post implantation loss

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

pre and post implantation loss

Remarks on result:

other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group G4, statistically significant reduction in mean pup (both male and female) weight per litter on PND 1, 4, 7 and 13 were noted. These changes are considered as test item-related due to increased pup mortalities, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

In group G4, reduced live birth index per litter (90.43%) was noted when compared with vehicle control group (99.09%). This noted change was statistically significant for the mean number of live born female pups and is considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no statistically significant differences observed in the sex ratio of pups between the treated groups and the vehicle control group.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

In group G4, reduced mean litter size at birth (8.11) was noted when compared with vehicle control group (10.73). This noted change is considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

In group G4, reduced number of live pups in either sex and pup survival index during lactation period were noted when compared with vehicle control group. These noted changes are statistically significant for number of survived male pups per litter during LD 1 to 4, number of survived female pups per litter during LD 1 to 4, 5 to 7 and 8 to 13, number of dead pups per litter during LD 1 to 4 and 5 to 7 and pup survival index between LD 1 to 4, 5 to 7 and 8 to 13 when compared with vehicle control group. These effects are considered as test item-related due to noted test item-related clinical signs, test item-related reduction in maternal body weights and feed consumption during gestation and lactation periods.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, the dead pups which were found dead by birth and during lactation period were noted with pale discoloration and red discoloration at nasal region from group G4 litters. No gross pathological changes were noted in any of the survived pups from group G4 and all pups from other dose group litters.

Skeletal malformations:

not specified

Visceral malformations:

no effects observed

Description (incidence and severity):

No visceral abnormalities were detected in any of the pups sacrificed.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Serum Thyroxine (T4) Levels: There were no changes noted in T4 levels of PND 13 pups in any of the tested dose groups when compared with vehicle control group. The obtained T4 levels of PND 13 pups are within the normal range of this species and strain. There were no changes noted in T4 levels of PND 4 pups from the tested dose groups G2 and G3 when compared with vehicle control group. In group G4, serum T4 levels of PND 4 pups was not conducted as there were no surplus pups remained from this dose level on PND 4. However, the obtained T4 levels of PND 4 pups from groups G2 and G3 are within the normal range of this species and strain.

Anogenital distance (AGD): In group G4, reduction in mean pup (both male and female) anogenital distance measurement (mm) and ratio per litter were noted. Changes in AGD ratio were found statistically significant in both sexes. These changes are considered as test item-related due to increased pup mortalities, decreased growth/weight of pups, occurrence of external changes in pups and decreased pup survival index per litter at this dose level. These effects can also correlate with test item-related maternal effects on litters during gestation and lactation periods.

Nipple retention in male pups: There were no occurrences of nipple/arealaes in any of the male pups examined on their PND 13 from any of the tested dose group litters and vehicle control group litters.

Details on embryotoxic / teratogenic effects:

The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in maternal animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

Remarks on result:

other: Effects observed at the highest tested dose of 9.5 mg/kg bw/day.

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

Description (incidence and severity):

Dead pups which were found dead by birth and during lactation period in Group 4 were noted with pale discoloration and red discoloration at nasal region.

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: anogenital distance

Description (incidence and severity):

Reduction in mean pup (both male and female) anogenital distance measurement (mm) and ratio per litter were noted in group G4. Changes in AGD ratio were found statistically significant.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Table 5. Summary of vaginal smear examination for determination of oestrus cyclicity

Determination of Oestrus Cyclicity during Pre-mating Treatment Period
Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		Females with Complete Regular Cycles	Females with at least one Irregular Oestrus Cycle	Average Length of Oestrous Cycle (Days)
G1, F & 0	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G2, F & 2	12	n	12	0	Mean	4.92
		%	100.00	0.00	±SD	0.19
					n	12
G3, F & 4.3	12	n	12	0	Mean	4.96
		%	100.00	0.00	±SD	0.14
					n	12
G4, F & 9.5	12	n	12	0	Mean	5.00
		%	100.00	0.00	±SD	0.00
					n	12

Table 6. Summary record of reproductive performance

Group, Sex & Dose   (mg/kg body weight/day)	No. of Males with Evidence of Mating (No. of Males used for Mating)	Male Mating Index (%)	No. of Males Capable of Impregnating a Female (No. of males used for Mating)	Male Fertiltiy Index (%)
G1, M & 0	11 (12)	91.7	11 (12)	91.7
G2, M & 2	11 (12)	91.7	11 (12)	91.7
G3, M & 4.3	11 (12)	91.7	11 (12)	91.7
G4, M & 9.5	11 (12)	91.7	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)		Cohabitation Record and Pre-coital Interval (Mean Time to Mating)				Gestational Length (duration of pregnancy)
		Pre-coital Interval (Days)		Concieving Days (1 to 5)	Concieving Days (More than 5 days)	Gestation Length (Days)
G1, F & 0	Mean	9.08	n	5	7	22.09
	±SD	6.05	%	41.67	58.33	0.54
	n	12				11
G2, F & 2	Mean	6.75	n	7	5	22.17
	±SD	5.33	%	58.33	41.67	0.39
	n	12				12
G3, F & 4.3	Mean	7.58	n	6	6	22.18
	±SD	5.70	%	54.55	54.55	0.60
	n	12				11
G4, F & 9.5	Mean	6.33	n	7	5	22.11
	±SD	5.38	%	58.33	41.67	0.33
	n	12				9

n: Number of dams

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)		Female Fertiltiy Index (%)
	No. of Females with Evidence of Mating (No. of Females used for Mating)	Female Mating Index (%)	No. of Females Confirmed as Fertile (No. of Females used for Mating)	Female Fertiltiy Index (%)
G1, F & 0	12 (12)	100.0	11 (12)	91.7
G2, F & 2	12 (12)	100.0	12 (12)	100.0
G3, F & 4.3	12 (12)	100.0	11 (12)	91.7
G4, F & 9.5	12 (12)	100.0	11 (12)	91.7

Group, Sex & Dose (mg/kg body weight/day)	Female Fecundity or Pregnancy Index (%)			Gestation Index (%)
	No. of Pregnant Females	No. of Females Confirmed with Mating	Female Fecundity or Pregnancy Index (%)	Females with Live Born Pups at Parturation	No. of Females with Evidence of Pregnancy	Gestation Index (%)
G1, F & 0	11	12	91.7	11	11	100.0
G2, F & 2	12	12	100.0	12	12	100.0
G3, F & 4.3	11	12	91.7	11	11	100.0
G4, F & 9.5	11	12	91.7	9	11	81.8

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)				Post-natal Loss (%)
		No. of Implantations	No. of Viable Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)	Total No. of Deaths/ Cannibalized during Lactation Period	Post-natal Loss (%)
G1, F & 0	Mean	10.82	10.64	0.18	1.82	0.00	0.00
	±SD	1.54	1.69	0.40	4.05	0.00	0.00
	n	11	11	11	11	11	11
G2, F & 2	Mean	10.67	10.42	0.25	2.68	0.00	0.00
	±SD	1.78	1.98	0.62	7.32	0.00	0.00
	n	12	12	12	12	12	12
G3, F & 4.3	Mean	11.00	10.82	0.18	1.53	0.00	0.00
	±SD	1.67	1.60	0.40	3.41	0.00	0.00
	n	11	11	11	11	11	11
G4, F & 9.5	Mean	12.56	7.44	5.11*	38.27*	4.67*	62.47*
	±SD	2.07	3.13	4.37	28.23	4.30	44.96
	n	9	9	9	9	9	9

n: Number of dams

* Statistically significant (P<0.05) change than the vehicle control group.

Table 7. Summary of delivery and litter observation record during lactation period

Group, Sex & Dose (mg/kg body weight/day)		Litter Size (No.)	Live Pups (No.)			Dead Pups (No.)			Cannibalized Pups (No.)				Sex Ratio (m/f) at Birth	Live Birth Index (%)
			Male	Female	Total	Male	Female	Total	Undetermined	Male	Female	Total
G1, F & 0	Mean	10.73	5.55	5.09	10.64	0.09	0.00	0.09	0.00	0.00	0.00	0.00	1.34	99.09
	±SD	1.62	2.11	1.64	1.69	0.30	0.00	0.30	0.00	0.00	0.00	0.00	1.01	3.02
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	10.50	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.08	0.00	0.08	1.15	99.40
	±SD	2.11	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.29	0.00	0.29	1.28	2.06
	n	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	10.91	5.09	5.73	10.82	0.09	0.00	0.09	0.00	0.00	0.00	0.00	0.99	99.30
	±SD	1.70	1.30	1.68	1.60	0.30	0.00	0.30	0.00	0.00	0.00	0.00	0.44	2.32
	n	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	8.11	4.00	3.44*	7.44	0.33	0.33	0.67	0.00	0.00	0.00	0.00	1.17	90.43
	±SD	2.67	1.73	2.24	3.13	0.71	0.71	1.32	0.00	0.00	0.00	0.00	0.86	21.83
	n	9	9	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

*: Statistically significant (P<0.05) change than the vehicle control group

Note: A total of 9 dams were considered for mean calculations of all end points/parameters at birth from group G4 as 1 out of 12 dams confirmed as non-pregnant; 2 out of 12 dams were confirmed with total litter loss [postimplantation loss].

Group, Sex & Dose (mg/kg body weight/day)		During LD 1 to 4									Sex Ratio (m/f) on LD 4	Pup Survival Index (%) LD 1 to 4
		Live Pups (No.)			Dead Pups (No.)			Cannibalized (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	5.55	5.09	10.64	0.00	0.00	0.00	0.00	0.00	0.00	1.34	100.00
	±SD	2.11	1.64	1.69	0.00	0.00	0.00	0.00	0.00	0.00	1.01	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.83	10.42	0.00	0.00	0.00	0.00	0.00	0.00	1.15	100.00
	±SD	0.90	2.08	1.98	0.00	0.00	0.00	0.00	0.00	0.00	1.28	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	5.73	10.82	0.00	0.00	0.00	0.00	0.00	0.00	0.99	100.00
	±SD	1.30	1.68	1.60	0.00	0.00	0.00	0.00	0.00	0.00	0.44	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	1.89*	1.22*	3.11*	2.11*	2.11*	4.22*	0.00	0.11	0.11	0.85	40.89*
	±SD	2.42	1.39	3.72	2.47	2.37	4.29	0.00	0.33	0.33	1.13	46.69
	n	9	9	9	9	9	9	9	9	9	9	9

n: Number of Dams; m/f: Male/Female;

*: Statistically significant (P<0.05) change than the vehicle control group;

Note: A total of 9 dams were considered for mean calculations of all end points/parameters during LD 1 to 4 from group G4 as 1 out of 12 dams confirmed as non-pregnant;

2 out of 12 dams were confirmed with total litter loss [postimplantation loss].

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	Pups Sacrificed for Blood Collection on LD 4 (No.)			Live Pups (No.) on LD 4 after Sacrificed for Blood Collection			During Lactation Day 5 to 7									Sex Ratio (m/f) on LD 7	Pup Survival Index (%) during LD 5 to 7
								Live Pups per litter (No.)			Dead Pups per litter (No.)			Cannibilized Pups per litter (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Total	Male	Female	Female
G1, F & 0	Mean	0.00	0.55	0.55	5.55	4.55	10.09	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	0.00	0.82	0.82	2.11	1.44	1.04	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	0.00	0.75	0.75	4.58	5.08	9.67	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.00	0.97	0.97	0.90	1.62	1.30	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	0.00	1.00	1.00	5.09	4.73	9.82	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	0.00	0.89	0.89	1.30	1.42	0.98	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	0.00	0.00	0.00	3.40	2.20	5.60	3.40	1.80*	5.20	0.00	0.40*	0.40*	0.00	0.00	0.00	1.70	77.50*
	±SD	0.00	0.00	0.00	2.30	1.10	3.21	2.30	1.30	3.42	0.00	0.55	0.55	0.00	0.00	0.00	1.20	43.66
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

n: Number of Dams; LD: Lactation Day; m/f: Male/Female

*: Statistically significant (P<0.05) change than the vehicle control group

Note: A total of 5 dams were considered for mean calculations of all end points/parameters during LD 5 to 7 from group G4 as 4 out of 9 dams were noted with total postnatal pup loss during LD 1 to 4.

Group, Sex & Dose (mg/kg body weight/day)	Animal No.	During Lactation Day 8 to 13									Sex Ratio (m/f) at LD 13	Pup Survival Index (%) during LD 8 to 13
		Live Pups (No.)			Dead Pups (No.)			Cannibalized (No.)
		Male	Female	Total	Male	Female	Total	Male	Female	Total
G1, F & 0	Mean	5.55	4.55	10.09	0.00	0.00	0.00	0.00	0.00	0.00	1.52	100.00
	±SD	2.11	1.44	1.04	0.00	0.00	0.00	0.00	0.00	0.00	1.14	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G2, F & 2	Mean	4.58	5.08	9.67	0.00	0.00	0.00	0.00	0.00	0.00	1.25	100.00
	±SD	0.90	1.62	1.30	0.00	0.00	0.00	0.00	0.00	0.00	1.26	0.00
	n	12	12	12	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	5.09	4.73	9.82	0.00	0.00	0.00	0.00	0.00	0.00	1.30	100.00
	±SD	1.30	1.42	0.98	0.00	0.00	0.00	0.00	0.00	0.00	0.95	0.00
	n	11	11	11	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	4.25	2.00*	6.25	0.00	0.25	0.25	0.00	0.00	0.00	2.38	97.22*
	±SD	1.50	0.82	1.71	0.00	0.50	0.50	0.00	0.00	0.00	0.95	5.56
	n	4	4	4	4	4	4	4	4	4	4	4

n: Number of Dams; LD: Lactation Day; m/f: Male/Female;

*: Statistically significant (P<0.05) change than the vehicle control group

Note: A total of 4 dams were considered for mean calculations of all end points/parameters during LD 8 to 13 from group G4 as 1 out of 5 dams were noted with total postnatal pup loss during LD 5 to 7.

Table 8. Summary record of pup observations during post-natal period

Group, Sex & Dose (mg/kg body weight/day)		At Birth (PND 1)	PND 1 to 4	Pups Sacrificed for Blood Collection on PND 4 (No.)*	PND 5 to 7^$	PND 8 to 13^$
G1, F & 0	No. of Dams^#	11	11	4	11	11
	No. of Live Pups	117	117	6	111	111
	Pup Observation/ No. of Pups observed	N/117	N/117	-	N/111	N/111
G2, F & 2	No. of Dams^#	12	12	5	12	12
	No. of Live Pups	125	125	9	116	116
	Pup Observation/ No. of Pups observed	N/125	N/125	-	N/116	N/116
G3, F & 4.3	No. of Dams^#	11	11	7	11	11
	No. of Live Pups	119	119	11	108	108
	Pup Observation/ No. of Pups observed	N/119	N/119	-	N/108	N/108
G4, F & 9.5	No. of Dams^#	9	9	0	9	9
	No. of Live Pups	67	28	0	26	25
	Pup Observation/ No. of Pups observed	P/28; HS/1; N/38	L/2; N/26	-	L, P/1; N/25	N/25

N: Normal; PND: Post-natal Day; #: Dams confirmed with littering; P: Pale colored; HS: Haemorrhagic spot; L: Lethargic

*: Pups selected for blood collection from dams with litter size of more than 10; $: Pups sacrificed on PND 4 were excluded

Table 9. Summary record of mean pup weight (g) per litter

Group, Sex & Dose (mg/kg body weight/day)		PND 1		PND 4		PND 7		PND 13
		Mean Pup Weight (g)		Mean Pup Weight (g)		Mean Pup Weight (g)		Mean Pup Weight (g)
		Male	Female	Male	Female	Male	Female	Male	Female
G1, F & 0	Mean	6.75	6.44	11.42	10.64	16.25	15.26	29.26	27.87
	±SD	0.09	0.17	0.36	0.20	0.47	0.35	0.51	0.82
	n	11	11	11	11	11	11	11	11
G2, F & 2	Mean	6.82	6.57	11.58	10.81	16.51	15.60	29.58	28.56
	±SD	0.14	0.16	0.37	0.19	0.37	0.40	0.25	0.40
	n	12	12	12	12	12	12	12	12
G3, F & 4.3	Mean	6.78	6.48	11.50	10.72	16.54	15.44	29.47	28.13
	±SD	0.12	0.18	0.32	0.23	0.30	0.33	0.33	0.65
	n	11	11	11	11	11	11	11	11
G4, F & 9.5	Mean	3.94*	3.73*	6.95*	5.70*	10.88*	9.11*	19.99*	17.88*
	±SD	0.89	0.80	1.79	1.91	3.28	2.75	4.47	4.49
	n	9	8	4	5	4	4	4	4

n: Number of litters

* Statistically significant (P<0.05) change than the vehicle control group.

Table 10. Summary record of mean pup ano-genital distance (AGD) measurement (mm) and ano-genital distance (AGD) ratio per litter on post-natal day 4

Group, Sex & Dose (mg/kg body weight/day)		Mean Male Pup AGD Measurement (mm)	Mean Female Pup AGD Measurement (mm)	Mean Male Pup AGD Ratio	Mean Female Pup AGD Ratio
G1, F & 0	Mean	5.43	2.69	2.41	1.22
	±SD	0.22	0.12	0.08	0.05
	n	11	11	11	11
G2, F & 2	Mean	5.42	2.77	2.39	1.25
	±SD	0.25	0.13	0.09	0.05
	n	12	12	12	12
G3, F & 4.3	Mean	5.34	2.70	2.37	1.23
	±SD	0.19	0.13	0.07	0.05
	n	11	11	11	11
G4, F & 9.5	Mean	4.07	1.78	2.14*	1.00*
	±SD	0.61	0.41	0.15	0.15
	n	4	5	4	5

n: Number of litters

* Statistically significant (P<0.05) change than the vehicle control group.

Table 11. Summary of male pup nipple/areolae retention (no.) record

Group, Sex & Dose (mg/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Post-natal Day 13
G1, F & 0	Mean	0.00
	±SD	0.00
	n	11
G2, F & 2	Mean	0.00
	±SD	0.00
	n	12
G3, F & 4.3	Mean	0.00
	±SD	0.00
	n	11
G4, F & 9.5	Mean	0.00
	±SD	0.00
	n	9

n: Number of litters

Conclusions:

The NOAEL of the test substance for maternal and developmental toxicity in rats by oral route was determined to be 4.3 mg/kg bw/day. The reproductive effects observed at the highest dose level of 9.5 mg/kg bw/day were considered as secondary non-specific effects occurred as a result of systemic toxicity in maternal animals due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance POLY-U by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 2, 4.3 and 9.5 mg/kg bw/d based on the results of two consecutive dose range finding studies performed with the same species for a period of 14 days. at doses of 50, 100 and 300 mg/kg bw /d and 2.7, 8.3 and 25 mg/kg bw /d respectively. The test item was administered to the main group males for a period of 37 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 5 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and for PND 4 (from available pups) and PND 13 pups.

In groups G2 and G3, there were no indication of test item-related effects in any of the parameters assessed for systemic or reproductive toxicity.

In group G4/G4R, the animals were noted with test item-related clinical signs of toxicity such as, lethargic, rough hair coat, wet perineum, chromodacryorrhea, hair thinning or blood stains around vagina, as well as test item-related reduction in body weight and feed consumption. Also, test item-related reduced motor activity assessments, effects on clinical pathology investigations and reduced thymus spleen and liver weights were found at the dose level of 9.5 mg/kg bw/d. During gross pathology, test item-related changes like wet pernineum (4/12 males and 7/12 females), wet pernineum along with blood stains around vagina (2/12 females) and uterus with resorptions/dead fetuses (2/12 females) were noted from group G4. Microscopically, test item-related changes were observed in spleen, thymus and bone marrow of femur in both sexes at this dose level. Thus, the investigations were extended to the lower dose and recovery groups for these organs. Effects were not observed in these groups. All these changes in thymus, spleen and femoral bone marrow were considered to be reversible, secondary effects of reduced food consumption and decreased body weight gain (Everds et.al., 2013). The high dose recovery group animals treated with 9.5 mg/kg bw/day were recovered from the systemic toxicity effects during recovery period.

In group G4, test item-related reduced gestation index, increased post-implantation loss, increased postnatal loss, reduced litter size, reduced live birth index per litter, increased number of dead pups at birth / during postnatal period and reduced pup survival index were noted. Also, test item-related external anomalies in pups, test item-related reduced mean pup weight and ano-genital distance ratio per litter in either sex were noted. However, there were no occurrences of nipples in male pups examined on PND 13 from all the litters. The pups which were found dead by birth and during postnatal period were noted with gross pathological changes like pale coloured and nose discoloration. No gross pathological changes were noted in any of the survived pups. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 pups.

However, these reproductive effects were considered as secondary non-specific effects occurred as a result of systemic toxicity due to absence of adverse effects or no effects noted on major reproductive end points at this dose.

Therefore, based on the above results discussed, the NOAEL of the test item POLY-U for maternal and developmental toxicity is considered to be 4.3 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

4.3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A key study is available with a Klimisch score of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a combined repeated dose toxicity study with the reproduction /developmental toxicity screening test (OECD 422) performed in rats with the test substance, there were no indication of test item-related effects in any of the parameters assessed for systemic or reproductive toxicity at the low and mid doses (2 and 4.3 mg/kg bw/day). At the high dose (9.5 mg/kg bw/day) some effects were seen like the reduction in the gestation index, in the live birth index and litter size, in the pup survival index, in the mean pup weight and in the mean anogenital ratio. These effects are considered as influenced by maternal toxicity because they were first seen at the same dose level where significant reduction in mean body weight gain of dams during the gestation period (from gestation day 7 to 20) and during the lactation period (from lactation day 4 to 13) also started to be observed. Furthermore, at this dose level there were no effects in other relevant reproductive parameters like the mating, fertility or pregnancy indices. Also, no gross pathological changes were noted in any of the survived pups, no occurrences of nipple retention in male pups or no changes noted in serum thyroxine (T4) hormone levels.

Based on these results, the reproductive effects observed at the dose level of 9.5 mg/kg bw/day are considered as secondary non-specific effects occurred as a result of systemic toxicity in parental animals and thus the substance should not be classified for reproductive toxicity in accordance with CLP Regulation (EC) no 1272/2008.