[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HanTac: WH

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 14-15 weeks

- Weight at study initiation: Males: 365 to 425 g, Females: 208 to 246 g

G1 (m/f): 391.33 ± 16.05 / 226.33 ± 09.83 g
G2 (m/f): 392.30 ± 14.45 / 226.51 ± 09.72 g
G3 (m/f): 390.10 ± 13.41 / 225.05 ± 09.66 g
G4 (m/f): 391.73 ± 14.84 / 223.27 ± 11.59 g

G1R (m/f): 379.56 ± 04.69 / 221.71 ± 01.52 g
G4R (m/f):381.16 ± 04.30 / 218.58 ± 03.11 g

- Fasting period before study: no
- Housing:
Pre-mating: Two rats of same sex were housed per cage except for the last animal in the recovery group which was housed singly in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper.
Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.
- Enrichment: Polycarbonate Rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. This was changed along with cage twice a week. The enrichment was provided both during pre-mating and post mating period for males and pre-mating period for females. For recovery groups enrichment was provided throughout the experimental period.

- Diet: Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, Wi 53744-4420 was provided ad libitum to the animals.
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: five days before start


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25°C
- Humidity (%): 65 - 68
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.1 % (w/v) Na CMC (medium viscosity) in Milli-Q® water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily prior to start of the treatment or prepared on need basis and used within the stability period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring using a magnetic stirrer.

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on the solubility and suspensibility test conducted for study no N3335 with the same test item.
- Concentration in vehicle: 0 / 11 / 33 / 100 mg//mL
- Amount of vehicle (if gavage): 10 mL/kg bwt/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item was determined quantitatively in dose formulations by UV/Vis spectrometry at 433 nm.
Calibration: linear regression line in the range from 5 to 30 µg/mL in DMSO.
Sample preparation: samples for analysis of active ingredient content and homogeneity were taken and diluted with the diluent to get the final concentration of about 15 µg/mL of the test item.

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during 2nd month (day 34) of treatment and were analysed in-house.
For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No. G13350. One set of samples was analysed for concentration (a.i) analysis.
Formulations were considered acceptable as mean results are within ± 15 % of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.
The unused back up samples were disposed as analysis results of the first set of samples were within the acceptable limits

Duration of treatment / exposure:

Males: The dose formulation was administered for a period of 49 days to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating period and until sacrifice.

Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed). The range of treatment duration was 52 to 56 days. The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours) for a period of 52 days.
The dose volume administered for each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups.

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The volume of dose administered was 10 mL/kg Bwt/day throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.

Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 10 mL/kg Bwt/day.

The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.

Frequency of treatment:

Daily once

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 Vehicle Control

Dose / conc.:

110 mg/kg bw/day (nominal)

Remarks:

G2 Low dose

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

G3 Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4 High dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R Vehicle Control recovery

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4R High dose recovery

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected in order to induce toxic effects at the highest dose, any dosage related response and no adverse effects at the mid and lowest dose.
- Grouping: Grouping was done by the method of body weight stratification and distribution.
- Rationale for selecting satellite groups: Satellit groups were used in the control and the top dose group for observation of reversibility, persistence or delayed occurrence of systemic toxic effects.
- Post-exposure recovery period in satellite groups: 14 day no treatment period

Positive control:

no

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment and recovery periods.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation and walking backwards.
On the days of detailed clinical examination, general clinical signs were not performed except on treatment Day 1

BODY WEIGHT:
i) Individual body weights of males were recorded initially and at weekly intervals thereafter. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on GD 0, 7, 14 and 20 and on LD 0, 4 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Cagewise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of lactation period.

ESTROUS CYCLICITY:
Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating period to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Sacrifice and pathology:

The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland was collected from all pups on LD 13 and examined microscopically. All gross lesions were examined from all the groups. The reproductive organs of any animals failing to mate were examined in all the dose groups.

Statistics:

ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data was entered retrospectively in ProvantisTM) and Clinical Chemistry data was analysed using Provantis built-in statistical tests.

The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights), body weight, food consumption, oestrous cycle length, hormone levels, anogenital distance, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and cont ol group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant.

Post-implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (Days) was analysed after suitable transformation (√ x + ½) of the data. Oneway ANOVA was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.

Z test was performed for testing the differences in proportions for mating and fertility indices.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs and mortalities observed both during the treatment and recovery periods at all the treated groups in both sexes.
However, the light to dark orange coloured faeces were observed in the treated group rats. The coloured faeces were attributed to the nature of the test item and not an adverse finding.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and net weight gains were unaffected by the treatment at all the doses tested in both sexes when compared to vehicle control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control group.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

eye examination: normal

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no indication for test item-related clinical and anatomic pathology changes in adult males and females at all the doses tested except for presence of light orange colored stomach/intestinal contests in all treated males and in one low dose female. This change in color of stomach/intestinal contents did not result in any gross/microscopic changes in respective tissues.

Neuropathological findings:

no effects observed

Description (incidence and severity):

No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: highest dose applied

Key result

Critical effects observed:

no

Conclusions:

Daily oral (gavage) administration of the test item to male and female Wistar rats at the dose levels of 110, 330 and 1000 mg/kg bw/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects.

The No Observed Adverse Effect Level (NOAEL) of the test item for general toxicity (repeated dose toxicity) in males and females is considered to be 1000 mg/kg bw/day.

Therefore, the test item has not to be classified as subacutely toxic (STOT RE) by the oral route according to Regulation (EC) No 1272/2008.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study was used to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was suspended in vehicle (0.1 % (w/v) Na CMC in Milli-Q® water} and administered by oral gavage at the dose levels of 110, 330 and 1000 mg/kg bwt/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. A concurrent vehicle control (G1) and a vehicle control recovery group (G1R) of rats received the vehicle alone. The dose volume administered was 10 mL/kg bw/day. The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 (for females). In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams. Animals in the recovery groups were not mated and consequently not used for assessment of reproduction/developmental toxicity.

The identity of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study G13350. Based on the results, the test item was found to be stable for up to 6 days at 1 and 100 mg/mL concentrations, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during Week 4 of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the nominal concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were examined for the appearance nipples/areolae on post-natal day (PND) 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group. For recovery groups, functional observation battery was done towards the end of recovery period. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams at termination on LD 13 and two pups per litter on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland was collected from all pups on LD 13 and examined microscopically. All gross lesions were examined from all the groups.

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

• There were no treatment-related clinical signs or mortality observed at any of the doses tested.

• No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.

• The body weights and food consumption were unaffected by the treatment at all the doses tested.

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

• Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses

• No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.

• The survival indices were not altered by the treatment at all the doses tested.

• There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size.

• There were no external abnormalities in live or dead pups in any of the groups.

• No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group.

• The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

• The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females.

• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females.

• Gross pathological examination of pups on LD 13 did not reveal any gross changes.

• There were no test item related gross and microscopic changes in both males and females. Microscopically in lungs, pigmented macrophages were observed at 1000 mg/kg Bwt/day in both sexes and considered as test item related non-adverse finding. This change showed complete recovery at the end of recovery period.

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

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

The No Observed Adverse Effect Level (NOAEL) of the test item for general toxicity (repeated dose toxicity) in males and females is considered to be 1000 mg/kg bwt/day.

Therefore, the test item has not to be classified as subacutely toxic (STOT RD) by the oral route according to Regulation (EC) No 1272/2008.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

EC Number:

255-965-2

EC Name:

[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Cas Number:

42844-93-9

Molecular formula:

C29H18N4NiO3

IUPAC Name:

Nickel, [1,3-dihydro-5,6-bis[[[2-(hydroxy-kO)-1-naphthalenyl]methylene]amino-kN]-2H-benzimidazol-2-onato(2-)]-, (SP-4-2)-

Test material form:

solid: nanoform

Specific details on test material used for the study:

- Stability under test conditions: The stability and homogeneity of the test item in the vehicle was established at 1 and 100 mg/mL under Advinus Study No. G13350. Based on the results, the test item was observed to form homogenous suspension in the vehicle and stable up to 5 days when stored at room temperature.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana

- Females (if applicable) nulliparous and non-pregnant: [Yes]
- Age at study initiation: 14 to 15 wks;
- Weight at study initiation: Males: 365.42 to 425.87 g; Females: 208.25 to 246.47 g

- Housing:
Pre-mating: Two rats of same sex were housed per cage except for the last animal in the recovery group which was housed singly in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper.

Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.

Enrichment: Polycarbonate Rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. This was changed along with cage twice a week. The enrichment was provided both during pre-mating and post mating period for males and pre-mating period for females. For recovery groups enrichment was provided throughout the experimental period.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, WI 53744-4420 was provided ad libitum to the animals. A sample of the diet was retained until finalization of the report.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: Start : 28 May 2017 End: 01 June 2017

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 and 25°C
- Humidity (%): 65 and 68 %
- Air changes (per hr): 12 - 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle
IN-LIFE DATES: From: 16 June 2017 To: 10 August 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: CMC, sodium salt

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The dose formulations were prepared daily prior to start of the treatment or prepared on need basis
and used within the stability period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring
using a magnetic stirrer.



VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the solubility test conducted for Study No. N3335 with same test item, the test item forms suspension in 0.1% (w/v) carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q water, while it could not be dissolved/suspended in Milli-Q water. Hence 0.1% carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q water was used as vehicle for dose formulation preparation.

Details on mating procedure:

- M/F ratio per cage: 1 : 1
- Length of cohabitation: 21 days
- Proof of pregnancy: [vaginal plug / 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): One per cage
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during 2nd month (day 34) of treatment and were analysed in-house.
For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No. G13350. One set of samples was analysed for concentration analysis.
Formulations were considered acceptable as mean results are within ± 15 % of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.
The unused back up samples were disposed as analysis results of the first set of samples were within the acceptable limits.

Duration of treatment / exposure:

Males: The dose formulations were administered to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for a period of 49 days which includes two weeks prior to mating and the treatment was continued during the mating and post mating period until sacrifice.
Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).The range of treatment duration was 52 to 56 days.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours) for a period of 52 days.
The dose volume administered for each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 10 mL/kg bwt.

Frequency of treatment:

Daily once

Details on study schedule:

- Age at mating of the mated animals in the study: 16 to 17 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 Vehicle control

Doses / concentrations 2

Dose / conc.:

110 mg/kg bw/day (nominal)

Remarks:

G2 Low dose

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

G3 Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4 High dose

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R Vehicle control recovery

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4R High dose recovery

No. of animals per sex per dose:

10 animals per sex per dose for main groups
5 animals per sex per dose for recovery groups

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were selected in order to induce toxic effects at the highest dose, any dosage related response and no adverse effects at the mid and lowest dose.
- Rationale for animal assignment (if not random): Grouping was done by the method of body weight stratification and distribution. On the day of randomization, based on the given temporary animal identification number, each female with normal oestrous cyclicity (4-5 day cycle) and adult males were weighed and the corresponding body weight was recorded. The body weight recorded was stratified in ascending order.
Statistical analysis and ensured that the weight variation was minimal and inter group variation did not exceed ± 20% of the mean body weight in each group and sex. Rats with extreme body weights were discarded. Grouping was done one day prior to initiation of the treatment.
- Post-exposure recovery period in satellite groups: 14 day no treatment period

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs, the observation for morbidity and mortality was carriedout once in the morning during holidays. All rats were observed for clinical signs once daily during the treatment and recovery periods.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment and recovery periods. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous
membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling aswell as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation and walking backwards.
On the days of detailed clinical examination, general clinical signs were not performed except ontreatment Day 1

BODY WEIGHT:
i) Individual body weights of males were recorded initially and at weekly intervals thereafter. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (tillmating confirmation) with males.
ii) All dams were weighed on GD 0, 7, 14 and 20 and on LD 0, 4 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Cagewise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of lactation period.

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Sperm parameters (parental animals):

Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [Yes]
- If yes, maximum of [8] pups/litter ([4]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, other:] Yes

GROSS EXAMINATION OF DEAD PUPS:
[Yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated [Section 9.10 in Main Report] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated [Section 9.10 in Main Report] were prepared for microscopic examination and weighed, respectively.

Statistics:

ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data was entered retrospectively in ProvantisTM) and Clinical Chemistry data was analysed using Provantis built-in statistical tests.
The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights), body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant.
Post-implantation loss (%), no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (Days) was analysed after suitable transformation (√ x + ½) of the data. One-way ANOVA was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.
Z test was performed for testing the differences in proportions for Day 4 survival index, mating and fertility indices.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group

Reproductive indices:

Reproductive Performance Data of Parents
a. Male mating index (%) = (Number of males with evidence of mating / Number of males cohabited) x 100
b. Male fertility index (%) = (Number of males siring a litter / Number of males cohabited) x 100
c. Female mating index (%) = (Number of females mated / Number of females cohabited) x 100
d. Female fertility index (%) = (Number of pregnant females (confirmed at necropsy) / Number of females used for mating) x 100
e. Mean number of implantations/group = (Total number of implantations / Total number of pregnant animals)
f. Post implantation loss (%) = (Number of implantations - Number of live pups / Number of implantations) x 100

Offspring viability indices:

Offspring viability indices
Litter Data
a. Mean litter size per group = (Total Number of pups / Total Number of littered animals)
b. Mean viable litter size = (No. of viable pups on Day 1 / No. of females littered)
c. Live birth index (%) = (No. of viable pups born (at first observation) / Total no. of pups born (at first observation)) X 100
d. Day 4 survival index (%) = (Number of viable pups on lactation Day 4 / Number of viable pups born) x 100
e. Sex Ratio (%) = (No. of male pups born / Total No. of pups born) x 100
f. Ano-genital Distance Ratio (mm/g1/3) = (Ano-genital distance / Cube root of body weight)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed both during the treatment and recovery periods at all the treated groups in both sexes. However, the light to dark orange coloured faeces were observed in the treated group rats. The coloured faeces were attributed to the nature of the test item and not an adverse finding.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities observed both during the treatment and recovery periods at all the treated groups in both sexes.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and net weight gains were unaffected by the treatment at all the doses tested in both sexes when compared to vehicle control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the hematology parameters of both males and females.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in the clinical chemistry parameters of both males and females.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological changes in the reproductive organs and other tissues/organ examined in both males and females.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation).
The calculated mean oestrous cycle length was 4.05 days in the vehicle control and 4.03 days in all the treated groups. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.
The test item was suspended in 0.1% carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q water and administered by oral gavage at the dose levels of 110, 330 and 1000 mg/kg Bwt/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. Concurrent control (G1) and a control recovery groups (G1R) of rats received vehicle alone. The dose volume administered was 10 mL/kg Bwt/day.
The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The dose formulations were administered once daily to specific groups of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females. In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams
The identity of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No. G13350. Based on the results, the test item was observed to form a homogeneous suspension in the vehicle and stable up to 5 days, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Days 1, and during 2nd (Day 34) month of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the nominal concentrations and overall relative standard deviation was less than 10%.
All rats were observed for clinical signs once daily. Body weight was recorded prior to start of treatment and weekly thereafter. Food consumption was recorded at weekly intervals except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All surviving male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each main group. For recovery groups, functional observation battery was done towards the end of recovery period. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 13 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.
Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. All gross lesions were examined in all the groups. The reproductive organs of any animals failing to mate were examined in all the dose groups. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland collected from LD 13 pups from all the groups was also evaluated. All gross lesions were examined from all the groups.
Under the experimental conditions employed, the following results were obtained:
• Clinical Signs and Mortalities: Light to dark orange coloured faeces was observed in the treated group rats, due to the nature (colour) of the test item. No mortalities occurred at any of the doses tested.

• Functional Observation Battery (FOB): No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.
• Body weights and Food Consumption: The body weights and food consumption were unaffected by the treatment at all the doses tested during pre-mating, gestation and lactation periods.
• Fertility Parameters: Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.
• Litter Data: The survival indices were not altered by the treatment at all the doses tested. There were no treatment-related effects on the mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.
• Clinical Pathology Investigations: The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females.
• Terminal Body Weights, Organ Weights and Organs Weight Ratios: There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females.
• Thyroid Hormone Levels: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration. No test item-related changes were observed in organ weights, gross and histopathology of thyroid gland of parental rats and pups.
• Gross pathology: Light orange coloured stomach/intestinal contents in all treated males and in one low dose female was considered as test-item-related. This change in color of stomach/intestinal contents did not result in any gross/microscopic changes in respective tissues. These changes were due to the nature of the test item and not adverse findings.
• Histopathology: There were no test item-related histopathological changes in the reproductive organs and other tissues/organ examined in both males and females.
No Observed Adverse Effect Level
Daily oral (gavage) administration of the test item to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 4 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects on any of the parameters evaluated. Hence, the No Observed Adverse Effect Level (NOAEL) of the test item for systemic, reproductive/developmental toxicity is considered to be 1000 mg/kg bw/day.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

other: Systemic toxicity

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

All the surviving pups were necropsied on lactation Day 13 and findings were recorded. Dead and moribund pups were examined for possible defects and/or cause of death.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter size, and number of dead pups at first observation.
There were no external abnormalities in live or dead pups in any of the groups.
No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean number and body weight of male and female (and total number) pups per litter were not affected by the treatment at all the doses tested.

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:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination of pups on LD 13 did not reveal any gross changes.
No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of pups.

Histopathological findings:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

To summarize, daily oral (gavage) administration of test item to Wistar rats at the dose levels 110, 330 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and approximately 4 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. Functional observations did not reveal any test item related changes at all the tested doses. The survival indices were not altered by the treatment. The test item administration did not reveal any treatment related changes in the hematology, coagulation, clinical chemistry, urinalysis, terminal fasting body weights/organ weights and histopathological changes in adult rats of both the sexes. Gross examination of pups on LD 13 did not reveal any gross changes. Further, the male and female reproductive organs did not reveal any changes. The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration. No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of parental rats and pups.

The clinical sign of light to dark orange coloured faeces was observed in all the treated groups. Gross pathological examination revealed light orange coloured contents in stomach/intestine in all treated males and in one low dose female. These changes were due to the nature of the test item and not adverse findings.

No Observed Adverse Effect Level
Daily oral (gavage) administration of the test item to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg bw/day for 2 weeks prior to mating, during mating, and 4 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects on any of the parameters evaluated. Hence, the No Observed Adverse Effect Level (NOAEL) of the test item for systemic, reproductive/developmental toxicity is considered to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by oral gavage in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.The test item,PV-Echtorange 6RLwas suspended in0.1% carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q water and administered by oral gavage at the dose levels of 110, 330 and 1000 mg/kg bw/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. Concurrent control (G1) and a control recovery groups (G1R) of rats received vehicle alone. The dose volume administered was 10 mL/kg Bwt/day.The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The dose formulations were administered once daily to specific groups of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females. In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of damsThe identity ofPV-Echtorange 6RLwas provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No. G13350. Based on the results, the test item was observed to form a homogeneous suspension in the vehicle and stable up to 5 days, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Days 1, and during 2^nd(Day 34) month of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the nominal concentrations and overall relative standard deviation was less than 10%.All rats were observed for clinical signs once daily. Body weight was recorded prior to start of treatment and weekly thereafter. Food consumption was recorded at weekly intervals except during the cohabitation period.Female rats were also weighed onGestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All surviving male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each main group. For recovery groups, functional observation battery was done towards the end of recovery period. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 13 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.

Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis. All gross lesions were examined in all the groups. The reproductive organs of any animals failing to mate were examined in all the dose groups. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland collected from LD 13 pups from all the groups was also evaluated. All gross lesions were examined from all the groups.

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

Clinical Signs and Mortalities: light to dark organge coloured faeces was observed in the treated group rats, due to the nature (colour) of the test item. No mortalities occured at any of the doses tested.

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

Body weights and Food Consumption: The body weights and food consumption were unaffected by the treatment at all the doses tested during pre-mating, gestation and lactation periods.

Fertility Parameters: Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses. No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.

Litter Data:The survival indices were not altered by the treatment at all the doses tested.There were no treatment-related effects on the mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Clinical Pathology Investigations: The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females.

Terminal Body Weights, Organ Weights and Organs Weight Ratios: There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females.

Thyroid Hormone Levels: The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration. No test item-related changes were observed in organ weights, gross and histopathology of thyroid gland of parental rats and pups.

Gross pathology: Light orange coloured stomach/intestinal contents in all treated males and in one low dose female was considered as test-item-related. This change in color of stomach/intestinal contents did not result in any gross/microscopic changes in respective tissues.These changes were due to the nature of the test item and not adverse findings.

Histopathology: There were no test item-related histopathological changes in the reproductive organs and other tissues/organ examined in both males and females.

No Observed Adverse Effect Level

Daily oral (gavage) administration of the test item to male and female Wistar rats at dose levels of 110, 330 and 1000 mg/kg bw/day for 2 weeks prior to mating, during mating, and 4 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects on any of the parameters evaluated. Hence, the No Observed Adverse Effect Level (NOAEL) of the test item for systemic, reproductive/developmental toxicity is considered to be 1000 mg/kg bw/day.