5,12-dihydroquino[2,3-b]acridine-7,14-dione

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The oral administration of test item over the time of the study, carried out according to the OECD guideline 422 (for main group males with a total of 31 days, for main group females ranging from 50 to 62 days and for recovery group animals with a total period of 52 days) did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.

Similar findings were observed in a supporting study according to OECD TG 443 in a close structural analogue substance, which also gave a NOAEL of 1000 mg/kg/d.

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Remarks:

In accordance with OECD Guideline for Testing of Chemicals, Number 443, “Extended One-Generation Reproductive Toxicity Study”, adopted on 25 June 2018.

Type of information:

experimental study

Remarks:

The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor.

Adequacy of study:

supporting study

Study period:

14 May 2021 to 02 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with the test item by oral gavage in Sprague Dawley Rats”.

The test item was administered continuously in graduated doses to three groups of males and females. The parental (P) generation animals were dosed for a defined
pre-mating period (10 weeks) and a two-week mating period. Treatment of the
P females was continued during pregnancy and lactation until termination following weaning of respective litters. The F1 offspring received further treatment with the test item from weaning till adulthood.
The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability and homogeneity of the test material in the vehicle under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item in dose formulations was established by the analytical investigation
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Triturated well in a mortar (grinding)

- Final concentration of a dissolved solid: Low dose-11.1, Mid dose-33.3 and High dose-100 mg/mL

FORM AS APPLIED IN THE TEST: Suspension

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of 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:

Animal Species: Rat (Rattus norvegicus)
Strain: Sprague Dawley
Justification for Selection of Species : Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Source of Supply : Hylasco Biotechnology India Pvt. Ltd,
Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

Animals were housed under standard laboratory conditions in an environmentally monitored, temperature-controlled room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.8 to 23.1oC, relative humidity
48 to 64%, with 12 hours light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
P Animals:
i. Pre-mating
Maximum of two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirmation on 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 during gestation and lactation periods.
Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
F1 Animals:
i. During Lactation period (postnatal period)
All F1 pups of both sexes were housed along with the dam.
ii. Post Weaning
Two animals of same sex and group per cage were housed.
Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rats throughout the experimental period. The contaminant analysis test reports for the feed are presented as Annexure 2.
A sample of each batch of the diet used during the study was retained under appropriate conditions (-20°C) and discarded on the day of finalization of the report.
Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% w/v

Details on exposure:

The test item formulations/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.
All the doses were administered in an equi-volume of 10 mL/kg with the concentration of 11.1, 33.3 and 100 mg/mL for 111, 333 and 1000 mg/kg body weight dose groups respectively. Vehicle was administered to vehicle control group at an equi-volume of 10 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight.
Parental (P) Generation Animals:
-The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration].
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration]. .
F1 Animals:Direct dosing by oral gavage
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Details on mating procedure:

Each P female was placed with a randomly selected, single and unrelated male from the same dose group (1:1 pairing) until evidence of mating. Day 0 of pregnancy was defined as the day on which mating evidence was confirmed (presence of sperm in vaginal smear).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in dose formulations was established by the Analytical Department of Bioneeds India Private Limited (Bioneeds study no.:
BIO-ANM 1717).
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

Duration of treatment / exposure:

Parental (P) Generation Animals
The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration]
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration
F1 Animals
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Frequency of treatment:

once daily

Details on study schedule:

Study Initiation Date: 14 May 2021
Experimental Starting Date: 14 May 2021
Parental (P) Generation
Acclimatization Date : Start: 14 May 2021; End: 19 May 2021
Treatment Date :
Males: Start: 20 May 2021; End: 22 August 2021
Females: Start: 20 May 2021; End: 20 September 2021
Cohabitation period: Start: 28 July 2021; End: 09 August 2021
Necropsy Date :
Males: Start: 16 August 2021; End: 23 August 2021
Females: Start: 10 September 2021; End: 21 September 2021
F1 Generation
Weaning Date: Start: 09 September 2021; End: 20 September 2021
Treatment Date :
Cohort 1A: Start: 09 September 2021; End: 03 December 2021
Cohort 1B: Start: 09 September 2021; End: 10 December 2021
Necropsy Date :
Cohort 1A: Start: 23 November 2021; End: 04 December 2021
Cohort 1B: Start: 30 November 2021; End: 11 December 2021
In-life End Date :11 December 2021
Histopathology Completion Date: 01 May 2022
Experimental Completion Date : 01 May 2022
Draft Report Submission Date : 30 May 2022
Study Completion Date: 02 August 2022

Dose / conc.:

0 mg/kg bw/day

Remarks:

Dose concentration = 0 mg/mL

Dose / conc.:

111 mg/kg bw/day

Remarks:

Dose concentration = 11.1 mg/mL

Dose / conc.:

333 mg/kg bw/day

Remarks:

Dose concentration = 33.3 mg/mL

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Dose concentration = 100 mg/mL

No. of animals per sex per dose:

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males + 80 females)]. Each cohort consisted of four groups and consisted of 20 males and 20 females per group.

Control animals:

yes, concurrent no treatment

Details on study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with by oral gavage in Sprague Dawley Rats”.

Parental animals: Observations and examinations:

Clinical Signs of Toxicity and Mortality/Morbidity
Detailed Clinical Examination
Body Weight
Feed Consumption
Oestrus Cyclicity
Reproductive Performance Evaluation
Delivery and Litter Observations
Uteri Observations
Clinical Pathology and Thyroid hormonal estimations
Thyroid Hormone Levels Estimation
Sperm Parameters
Gross Pathology
Histopathology

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored daily for a period of 2-weeks during pre-mating period before initiation of cohabitation.
During cohabitation period until evidence of mating.
At termination (on the day of necropsy) to correlate with histopathology of reproductive organs.

Sperm parameters (parental animals):

Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. Samples for evaluating sperm morphology was taken from the suspension used for the sperm motility. Sperm sample was examined as fixed and 200 spermatozoa per sample were classified as either normal (both head and mid-piece/tail appear normal) or abnormal. Another testis reserved for spermatid head counts was stored in -20ºC from each animal and subjected to evaluation for homogenization resistance spermatid head counts to calculate daily sperm production.

Litter observations:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated.
The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated

Postmortem examinations (parental animals):

All the parental males were sacrificed after completion of mating procedure. Males were randomized throughout all dose groups and restricted to maximum of 20 animals per day for necropsy.
The females not confirmed with mating were sacrificed after 25 days from the day of termination of cohabitation process. The females confirmed with mating but not littered were sacrificed 25 days after confirmation of mating. All the littered females were sacrificed on lactation day 22. Organ Weight and Tissue Preservation
The organs such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] from all P animals were collected, weighed, and preserved.

Histopathology
Histopathological examination was conducted on all the tissues collected from the vehicle control (G1) and high dose (G4) group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.
Additionally, reproductive organs of all animals suspected of reduced fertility (those that failed to conceive or sire from all the dose groups) were subjected to histopathological examination.

Postmortem examinations (offspring):

Necropsy and Gross Pathology

The animals were euthanized using deep Isoflurane anaesthesia followed by exsanguination and subjected to gross pathological examination. Gross pathological examination was performed on all the animals sacrificed.
Cohort 1A and 1B Males and Females: The cohort 1A animals were sacrificed on PND 96 and the cohort 1B animals were sacrificed on PND 103.

Cohort 1A: The organs and tissues such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] of all high dose and control animals were examined for histopathology.

Cohort 1A Females (Ovarian Follicular and Corpora luteal Assessments):
Histopathological examination of ovaries including quantitative evaluation of primordial and small growing follicles, as well as corpora lutea was conducted for all control and high dose animals.

Cohort 1B: The reproductive and endocrine tissues were processed to the block stage.

Organ-typic Development:
C1A Males: Caput, corpus and cauda of the epididymis and the vas deferens were examined for appropriate organ-typic development.
C1A Females: Ovary with oviduct, uterus and vagina were examined for appropriate organ-typic development.

Statistics:

The raw data was subjected to computer statistical processing. The data was subjected to various statistical analyses using SPSS software version 27.

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05) indicated by the aforementioned tests.

The statistical analysis was followed to the parameters as mentioned below table.
Parameter
Type of Analysis - One-way ANOVA with Dunnett's post-test
• Bodyweight (weekly/gestation/lactation)
• Percent Change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Mean oestrus cycle length
• Absolute/relative organ weights
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum thyroid hormonal values
• Occurrence of postnatal developmental landmarks of F1 pups
• Responses for sensory reflexes of F1 pups
• Sexual maturation time points of F1 animals
• Splenic lymphocyte sub-populations
• Sperm motility/morphological changes/daily sperm production
• Ovarian follicular count (Independent sample T - test) Parametric -
Type of Analysis - Non-parametric - Kruskal-Wallis followed by the Mann-Whitney
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter at birth and during the lactation period
• Litter size at birth and during the lactation period
• Post-implantation loss/litter
• Postnatal loss/litter
Type of Analysis - Cross Tabs - Frequency comparison Chi-square test/ Fischer's Exact Test
• Reproductive indices
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions

Reproductive indices:

The following reproductive performance indices were calculated for all P animals.

Mating and Fertility Index :
The male / female mating, and fertility indices were calculated as mentioned below:
Male mating index (%) = No. of males with confirmed mating/ Total no. of males cohabited X 100

Female mating index (%)= No. of sperm-positive females/Total no. of females cohabited X 100

Male fertility index (%)=No. of males impregnating a female/Total no. of males cohabited X 100

Female fertility index (%)= No. of females with evidence of implantation sites/No. of sperm-positive females X 100

Cohabitation Record and Copulatory Interval (Pre-coital Interval)

The day of initiation of mating and day of confirmation of mating were recorded for each female, and the pre-coital interval was calculated as mentioned below:

Pre-coital interval (days)=Date of confirmation of mating – Date of initiation of cohabitation

Gestation Length: The day of confirmation of mating and day of parturition were recorded for each female and the gestation length per litter was calculated as mentioned below:

Gestation length (days)=Date of parturition – Date of evidence of mating (GD 0)

Gestation Index
The gestation index (%) per group was calculated as mentioned below:
Gestation index (%)=No. of females with live born/No. of females with evidence of pregnancy X 100

Parturition Index
The parturition index (%) per group was calculated as mentioned below:
Parturition index (%)= No. of females littered/No. of females with evidence of pregnancy X 100

Female Fecundity or Pregnancy Index
The pregnancy index (%) per group was calculated as mentioned below:
Pregnancy index (%) = No. of females evident of presence of live/dead fetuses/pups /No. of females with evidence of matingX 100

Offspring viability indices:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated (litter as a unit) as mentioned below:
Sex ratio (m/f)= No. of male offspring/Number of female offspring
Live birth index (%) per litter=No. of pups born alive/Total no. of pups bornX 100

The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated (litter as a unit) as mentioned below:

Pup survival index (%) on LD 4/7/14/21 =Total no. of live pups on LD 4/7/14/21/No. of pups born/4/7/14X 100

The sex ratio (m/f) on LD 4, 7, and 13 was calculated per litter, as mentioned below:
Sex ratio (m/f) on LD 4/7/14/21=No. of male offspring on LD 4/7/14/21/No. of female offspring on LD 4/7/14/21

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Dark pink coloured faeces were noted in all the tested dose group animals of both sexes. This coloration is due to coloured nature of the test item but not an adverse sign.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In group G3, statistically significant reduction in percent change in mean gestational body weight gain during GD 7 to 14 was noted when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence and there were no such incidences noted during other phases of the gestation such as during GD 0 to 7 and 14 to 20 in the same dose level.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

In group G2 males, statistically significant reduction in mean feed consumption was noted during week 1 when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence for mean feed consumption and there were no such incidences noted throughout the experimental period at the same dose level.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant increase in mean haematocrit value in all the tested dose group males and statistically significant increase in mean absolute lymphocytes of group G2 males were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant decrease in mean total protein levels of group G4 females were noted when compared with vehicle control group.This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and also similar changes did not occur in other sex of same dose level.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The few microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A mean oestrus cycle length of 4.76, 4.91, 4.95 and 4.92 days was noted from groups G1, G2, G3 and G4 respectively. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups.
However, statistically significant increase in mean oestrus cycle length was noted in all the tested dose groups when compared with vehicle control group. This noted change is not considered as test item related as the obtained mean values are well within the
in-house historical control range and no irregularities in oestrus cycle were noted in any of the tested dose groups.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Effect level:

ca. 1 000

Based on:

test mat.

Remarks:

test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other:

Remarks on result:

other: test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The dark pink coloured faeces were noted in all the testing dose group animals of both sexes. This coloration is due to coloured nature of the test item but not any adverse signs.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
The noted non-toxicological and statistically significant increase in mean body weight on PND 42 in group G2 females when compared with vehicle control group .

A statistically significant decrease in mean body weights on PND 63, 70, 77, 84 and 91 and statistically significant decrease in percent change in mean body weight gain with respect to postnatal day (PND) 21 on PND 63, 70, 77, 84 and 91 in groups G3 and G4 males were noted when compared with vehicle control group.
These changes are not considered as adverse as there were no other systemic toxicity effects noted in any of these tested dose groups but are considered as stress induced effects due to test item administration.
Cohort 1B (C1B)
The noted non-toxicological and statistically significant increase in mean body weights on PND 77 and 84 in group G2-C1B females; increase in mean body weights on
PND 77, 84, 91 and 98 in group G3-C1B females; increase in mean body weights on PND 84, 91 and 98 in group G4-C1B females; increase in percent change in mean
body weight gain with respect to postnatal day (PND) 21 on PND 84 in group
G3-C1B females when compared with vehicle control group are considered as incidental.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
A statistically significant decrease in mean feed consumption during week 4 in group G3-C1A males when compared with vehicle control group is considered as incidental.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in mean haemoglobin, haematocrit, and prothrombin time levels in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in mean glucose, creatinine, cholesterol, and albumin levels of group G4 males; statistically significant decrease in mean creatinine levels in all the tested dose group females; statistically significant decrease in mean albumin levels in group G3 females was noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)

In group G2-C1A,
- decrease in mean absolute weight of liver and increase in relative weight of brain (males).
- increase in mean absolute and relative weight of liver and increase in absolute weight of iliac lymph nodes (females).

In group G3-C1A,
- decrease in mean terminal body weight (males).
- increase in mean relative weight of adrenals, kidneys and thyroid along with parathyroid (males).
- increase in mean relative weight of liver (females).

In group G4-C1A,
- decrease in mean terminal body weight (males).
- decrease in mean absolute weight of epididymides, heart, brain, liver (males); increase in mean relative weight of adrenals, testes, kidneys, prostate and thyroid along with parathyroid (males).
- increase in mean absolute and relative weight of thymus (females).

Cohort 1B (C1B)

In group G2-C1B,
- decrease in mean relative weight of mandibular lymph nodes (females).
In group G3-C1B,
- decrease in mean relative weight of adrenals and thyroid along with parathyroid (males).
- increase in mean absolute weight of spleen (females).
In group G4-C1B,
- increase in mean terminal body weight (females).
- decrease in mean absolute and relative weight of adrenals (males).
- decrease in mean relative weight of iliac and mandibular lymph nodes (females).

These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also neither gross pathological changes nor microscopic changes were noted in any of these organs in all the tested dose group animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In test-item administered group of animals of both the sexes, the contents of gastrointestinal tract were found to be pink in colour which could be attributed to physical appearance of the test item (pink in colour).
A single incidence of small sized testis was observed in G3-C1A group male rat and was microscopically correlated with moderate atrophy of tubules. This change was considered as an isolated spontaneous finding in rats.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few of the microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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.
Quantitative ovarian follicular assessment (primordial and primary follicles) in all parental and C1A females of control and high dose groups did not reveal any test item related variations. Similarly, quantitative evaluation of corpora luteal count in
C1A females of control and high dose group did not reveal any test item related variations.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Splenic Lymphocyte Sub-populations statistically significant increase in mean T cytotoxic (CD8+) cells populations in group G4-C1A males; increase in mean T cytotoxic (CD8+) cells populations in group G3-C1A females; decrease in T "helper" (CD4+) cells populations in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment as the obtained mean value is within in-house historical control range of same species and strain.

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day)
when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to
102 (for C1B animals) under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Generation:

other: cohort 1A and cohort 1B

Effect level:

>= 1 000

Based on:

test mat.

Remarks:

the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: Splenic Lymphocyte Subpopulation Analysis

Remarks on result:

other: the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods up to weaning of F1 animals (P females) as well as when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to 102 (for C1B animals) under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Executive summary:

The objective of this Extended One-Generation Reproductive Toxicity (EOGRT) Study in Sprague Dawley Rats was to evaluate the effects of test item as a result of pre and postnatal exposure on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females, young and adult offspring.
The study was conducted to serve as a test for reproductive endpoints that required the interaction of males with females, females with conceptus, females with offspring and the F1 generation before and after sexual maturity. This study was also conducted to evaluate the spermatogenesis for males and oestrous cycles, follicle counts/oocyte maturation and ovarian integrity (histopathology) for females.

This EOGRT study was also conducted to evaluate gonadal function, the oestrus cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition and lactation. Furthermore, the present EOGRT study was conducted to provide detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, and was expected to identify specific target organs in the offspring. In addition, this study provided and/or confirmed information about the effects of Hostaperm Pink E on the integrity and performance of the adult male and female reproductive systems.

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males
+ 80 females)]. Each cohort consisted of four groups and consisted of 20 males and
20 females per group. The animals in G1 (P generation) and G1-C1A/G1-C1B
(F1 generation) groups were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], the animals in G2 (P generation) and G2-C1A/G2-C1B (F1 generation),
G3 (P generation) and G3-C1A/G3-C1B (F1 generation), and G4 (P generation) and G4-C1A/G4-C1B (F1 generation) groups were administered with Hostaperm Pink E at the dose levels of 111, 333 and 1000 mg/kg body weight/day respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115 at the concentrations of 10.0 mg/mL and
100.0 mg/mL. Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1, 8, 16 and 26 of the treatment periods and the mean results were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

All the P generation animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) was recorded once weekly for all the P animals. The assessment for haematology, clinical chemistry, urinalysis, and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each P generation group at termination. Sperm parameters (motility, morphology, and sperm concentration/daily sperm production) were estimated for all P males. Oestrus cyclicity evaluation was determined during pre-mating and cohabitation period for all P females.
The body weights and feed consumption were recorded during gestation (Gestation Day 0, 7, 14 and 20) and during lactation (Lactation Day 1, 4, 7, 14 and 21) for all the
P females. The gross pathology and organ weighing was performed on the day of termination for all the P animals. Histopathological examination was conducted on all the tissues collected from the P generation vehicle control and high dose group animals. All the P males and females were evaluated for reproductive performance or indices such as, mating and fertility index (for P males and females) and pre-coital interval, gestation length, fecundity index, gestation index, post-implantation loss and postnatal loss (for females). All P females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter).

All the P males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups. However, in test-item administered group animals of both the sexes, the faecal matter was noted with ‘pink’ in colour which could be attributed to physical appearance of the test item (pink in colour). There were no test item-related histopathological findings noted in high dose parental animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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 of this strain and age.

The F1 generation pups were observed once daily for external examinations and
twice daily for mortalities till termination, weighed individually on postnatal day (PND) 1, 4, 7, 14 and 21, observed for occurrences of postnatal developmental landmarks, observed for responses towards to sensory reflexes during postnatal period, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13, observed for gross pathological observations at termination. The assessment for serum Thyroxine (T4) levels was conducted for PND 4 pups and assessment for serum Thyroxine (T4) and Thyroid Stimulating Hormone levels was conducted for PND 21 pups.

There were no test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels in F1 generation pups. No gross pathological changes noted in of the any of the F1 pups of both sexes from all the tested dose group litters.

All the Cohort 1A (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1A animals were evaluated for occurrence of sexual maturation (for balanopreputial separation in C1A males and for vaginal patency in C1A females). All C1A females were evaluated for mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells. All C1A females were evaluated for oestrus cyclicity from PND 75 to until sacrifice. The assessment for haematology, clinical chemistry, urinalysis and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each C1A group at termination. Sperm parameters (motility, morphology and daily sperm production) were estimated for all C1A males. The gross pathology and organ weighing were performed on the day of termination for all the C1A animals. Histopathological examination was conducted on all the tissues collected from the
C1A vehicle control and high dose group animals. The flow cytometric analysis of splenic samples for assessment of splenic lymphocyte sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes was conducted for randomly selected males and females from each C1A group.

All the C1A males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups when compared with vehicle control group, except slight reduction in mean body weight and percent change in mean body weight gain in all the tested dose group males. There were no changes noted in sub-populations of splenic lymphocytes such as, T "helper" (CD4+) cells,
T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes at any of the tested dose groups when compared with vehicle control group. There were no test item-related histopathological findings noted in high dose C1A animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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.

All the Cohort 1B (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1B animals were evaluated for occurrence of sexual maturation (for balano-preputial separation in C1B males and for vaginal patency in C1B females). The gross pathology and organ weighing were performed on the day of termination for all the C1B animals.

All the C1B males and females did not reveal any test item related changes in systemic and reproductive endpoints, except reduction in mean body weight and percent change in mean body weight gain in all the tested dose group of both sexes.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Remarks:

In accordance with OECD Guideline for Testing of Chemicals, Number 443, “Extended One-Generation Reproductive Toxicity Study”, adopted on 25 June 2018.

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

14 May 2021 to 02 August 2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Read across

Justification for type of information:

Please see read across justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with the test item by oral gavage in Sprague Dawley Rats”.

The test item was administered continuously in graduated doses to three groups of males and females. The parental (P) generation animals were dosed for a defined
pre-mating period (10 weeks) and a two-week mating period. Treatment of the
P females was continued during pregnancy and lactation until termination following weaning of respective litters. The F1 offspring received further treatment with the test item from weaning till adulthood.
The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability and homogeneity of the test material in the vehicle under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item in dose formulations was established by the analytical investigation
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Triturated well in a mortar (grinding)

- Final concentration of a dissolved solid: Low dose-11.1, Mid dose-33.3 and High dose-100 mg/mL

FORM AS APPLIED IN THE TEST: Suspension

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of 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:

Animal Species: Rat (Rattus norvegicus)
Strain: Sprague Dawley
Justification for Selection of Species : Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Source of Supply : Hylasco Biotechnology India Pvt. Ltd,
Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

Animals were housed under standard laboratory conditions in an environmentally monitored, temperature-controlled room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.8 to 23.1oC, relative humidity
48 to 64%, with 12 hours light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
P Animals:
i. Pre-mating
Maximum of two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirmation on 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 during gestation and lactation periods.
Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
F1 Animals:
i. During Lactation period (postnatal period)
All F1 pups of both sexes were housed along with the dam.
ii. Post Weaning
Two animals of same sex and group per cage were housed.
Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rats throughout the experimental period. The contaminant analysis test reports for the feed are presented as Annexure 2.
A sample of each batch of the diet used during the study was retained under appropriate conditions (-20°C) and discarded on the day of finalization of the report.
Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% w/v

Details on exposure:

The test item formulations/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.
All the doses were administered in an equi-volume of 10 mL/kg with the concentration of 11.1, 33.3 and 100 mg/mL for 111, 333 and 1000 mg/kg body weight dose groups respectively. Vehicle was administered to vehicle control group at an equi-volume of 10 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight.
Parental (P) Generation Animals:
-The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration].
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration]. .
F1 Animals:Direct dosing by oral gavage
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Details on mating procedure:

Each P female was placed with a randomly selected, single and unrelated male from the same dose group (1:1 pairing) until evidence of mating. Day 0 of pregnancy was defined as the day on which mating evidence was confirmed (presence of sperm in vaginal smear).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in dose formulations was established by the Analytical Department of Bioneeds India Private Limited (Bioneeds study no.:
BIO-ANM 1717).
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

Duration of treatment / exposure:

Parental (P) Generation Animals
The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration]
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration
F1 Animals
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Frequency of treatment:

once daily

Details on study schedule:

Study Initiation Date: 14 May 2021
Experimental Starting Date: 14 May 2021
Parental (P) Generation
Acclimatization Date : Start: 14 May 2021; End: 19 May 2021
Treatment Date :
Males: Start: 20 May 2021; End: 22 August 2021
Females: Start: 20 May 2021; End: 20 September 2021
Cohabitation period: Start: 28 July 2021; End: 09 August 2021
Necropsy Date :
Males: Start: 16 August 2021; End: 23 August 2021
Females: Start: 10 September 2021; End: 21 September 2021
F1 Generation
Weaning Date: Start: 09 September 2021; End: 20 September 2021
Treatment Date :
Cohort 1A: Start: 09 September 2021; End: 03 December 2021
Cohort 1B: Start: 09 September 2021; End: 10 December 2021
Necropsy Date :
Cohort 1A: Start: 23 November 2021; End: 04 December 2021
Cohort 1B: Start: 30 November 2021; End: 11 December 2021
In-life End Date :11 December 2021
Histopathology Completion Date: 01 May 2022
Experimental Completion Date : 01 May 2022
Draft Report Submission Date : 30 May 2022
Study Completion Date: 02 August 2022

Dose / conc.:

0 mg/kg bw/day

Remarks:

Dose concentration = 0 mg/mL

Dose / conc.:

111 mg/kg bw/day

Remarks:

Dose concentration = 11.1 mg/mL

Dose / conc.:

333 mg/kg bw/day

Remarks:

Dose concentration = 33.3 mg/mL

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Dose concentration = 100 mg/mL

No. of animals per sex per dose:

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males + 80 females)]. Each cohort consisted of four groups and consisted of 20 males and 20 females per group.

Control animals:

yes, concurrent no treatment

Details on study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with by oral gavage in Sprague Dawley Rats”.

Parental animals: Observations and examinations:

Clinical Signs of Toxicity and Mortality/Morbidity
Detailed Clinical Examination
Body Weight
Feed Consumption
Oestrus Cyclicity
Reproductive Performance Evaluation
Delivery and Litter Observations
Uteri Observations
Clinical Pathology and Thyroid hormonal estimations
Thyroid Hormone Levels Estimation
Sperm Parameters
Gross Pathology
Histopathology

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored daily for a period of 2-weeks during pre-mating period before initiation of cohabitation.
During cohabitation period until evidence of mating.
At termination (on the day of necropsy) to correlate with histopathology of reproductive organs.

Sperm parameters (parental animals):

Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. Samples for evaluating sperm morphology was taken from the suspension used for the sperm motility. Sperm sample was examined as fixed and 200 spermatozoa per sample were classified as either normal (both head and mid-piece/tail appear normal) or abnormal. Another testis reserved for spermatid head counts was stored in -20ºC from each animal and subjected to evaluation for homogenization resistance spermatid head counts to calculate daily sperm production.

Litter observations:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated.
The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated

Postmortem examinations (parental animals):

All the parental males were sacrificed after completion of mating procedure. Males were randomized throughout all dose groups and restricted to maximum of 20 animals per day for necropsy.
The females not confirmed with mating were sacrificed after 25 days from the day of termination of cohabitation process. The females confirmed with mating but not littered were sacrificed 25 days after confirmation of mating. All the littered females were sacrificed on lactation day 22. Organ Weight and Tissue Preservation
The organs such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] from all P animals were collected, weighed, and preserved.

Histopathology
Histopathological examination was conducted on all the tissues collected from the vehicle control (G1) and high dose (G4) group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.
Additionally, reproductive organs of all animals suspected of reduced fertility (those that failed to conceive or sire from all the dose groups) were subjected to histopathological examination.

Postmortem examinations (offspring):

Necropsy and Gross Pathology

The animals were euthanized using deep Isoflurane anaesthesia followed by exsanguination and subjected to gross pathological examination. Gross pathological examination was performed on all the animals sacrificed.
Cohort 1A and 1B Males and Females: The cohort 1A animals were sacrificed on PND 96 and the cohort 1B animals were sacrificed on PND 103.

Cohort 1A: The organs and tissues such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] of all high dose and control animals were examined for histopathology.

Cohort 1A Females (Ovarian Follicular and Corpora luteal Assessments):
Histopathological examination of ovaries including quantitative evaluation of primordial and small growing follicles, as well as corpora lutea was conducted for all control and high dose animals.

Cohort 1B: The reproductive and endocrine tissues were processed to the block stage.

Organ-typic Development:
C1A Males: Caput, corpus and cauda of the epididymis and the vas deferens were examined for appropriate organ-typic development.
C1A Females: Ovary with oviduct, uterus and vagina were examined for appropriate organ-typic development.

Statistics:

The raw data was subjected to computer statistical processing. The data was subjected to various statistical analyses using SPSS software version 27.

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05) indicated by the aforementioned tests.

The statistical analysis was followed to the parameters as mentioned below table.
Parameter
Type of Analysis - One-way ANOVA with Dunnett's post-test
• Bodyweight (weekly/gestation/lactation)
• Percent Change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Mean oestrus cycle length
• Absolute/relative organ weights
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum thyroid hormonal values
• Occurrence of postnatal developmental landmarks of F1 pups
• Responses for sensory reflexes of F1 pups
• Sexual maturation time points of F1 animals
• Splenic lymphocyte sub-populations
• Sperm motility/morphological changes/daily sperm production
• Ovarian follicular count (Independent sample T - test) Parametric -
Type of Analysis - Non-parametric - Kruskal-Wallis followed by the Mann-Whitney
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter at birth and during the lactation period
• Litter size at birth and during the lactation period
• Post-implantation loss/litter
• Postnatal loss/litter
Type of Analysis - Cross Tabs - Frequency comparison Chi-square test/ Fischer's Exact Test
• Reproductive indices
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions

Reproductive indices:

The following reproductive performance indices were calculated for all P animals.

Mating and Fertility Index :
The male / female mating, and fertility indices were calculated as mentioned below:
Male mating index (%) = No. of males with confirmed mating/ Total no. of males cohabited X 100

Female mating index (%)= No. of sperm-positive females/Total no. of females cohabited X 100

Male fertility index (%)=No. of males impregnating a female/Total no. of males cohabited X 100

Female fertility index (%)= No. of females with evidence of implantation sites/No. of sperm-positive females X 100

Cohabitation Record and Copulatory Interval (Pre-coital Interval)

The day of initiation of mating and day of confirmation of mating were recorded for each female, and the pre-coital interval was calculated as mentioned below:

Pre-coital interval (days)=Date of confirmation of mating – Date of initiation of cohabitation

Gestation Length: The day of confirmation of mating and day of parturition were recorded for each female and the gestation length per litter was calculated as mentioned below:

Gestation length (days)=Date of parturition – Date of evidence of mating (GD 0)

Gestation Index
The gestation index (%) per group was calculated as mentioned below:
Gestation index (%)=No. of females with live born/No. of females with evidence of pregnancy X 100

Parturition Index
The parturition index (%) per group was calculated as mentioned below:
Parturition index (%)= No. of females littered/No. of females with evidence of pregnancy X 100

Female Fecundity or Pregnancy Index
The pregnancy index (%) per group was calculated as mentioned below:
Pregnancy index (%) = No. of females evident of presence of live/dead fetuses/pups /No. of females with evidence of matingX 100

Offspring viability indices:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated (litter as a unit) as mentioned below:
Sex ratio (m/f)= No. of male offspring/Number of female offspring
Live birth index (%) per litter=No. of pups born alive/Total no. of pups bornX 100

The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated (litter as a unit) as mentioned below:

Pup survival index (%) on LD 4/7/14/21 =Total no. of live pups on LD 4/7/14/21/No. of pups born/4/7/14X 100

The sex ratio (m/f) on LD 4, 7, and 13 was calculated per litter, as mentioned below:
Sex ratio (m/f) on LD 4/7/14/21=No. of male offspring on LD 4/7/14/21/No. of female offspring on LD 4/7/14/21

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Dark pink coloured faeces were noted in all the tested dose group animals of both sexes. This coloration is due to coloured nature of the test item but not an adverse sign.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In group G3, statistically significant reduction in percent change in mean gestational body weight gain during GD 7 to 14 was noted when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence and there were no such incidences noted during other phases of the gestation such as during GD 0 to 7 and 14 to 20 in the same dose level.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

In group G2 males, statistically significant reduction in mean feed consumption was noted during week 1 when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence for mean feed consumption and there were no such incidences noted throughout the experimental period at the same dose level.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant increase in mean haematocrit value in all the tested dose group males and statistically significant increase in mean absolute lymphocytes of group G2 males were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant decrease in mean total protein levels of group G4 females were noted when compared with vehicle control group.This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and also similar changes did not occur in other sex of same dose level.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The few microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A mean oestrus cycle length of 4.76, 4.91, 4.95 and 4.92 days was noted from groups G1, G2, G3 and G4 respectively. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups.
However, statistically significant increase in mean oestrus cycle length was noted in all the tested dose groups when compared with vehicle control group. This noted change is not considered as test item related as the obtained mean values are well within the
in-house historical control range and no irregularities in oestrus cycle were noted in any of the tested dose groups.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Effect level:

ca. 1 000

Based on:

test mat.

Remarks:

test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other:

Remarks on result:

other: test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

The dark pink coloured faeces were noted in all the testing dose group animals of both sexes. This coloration is due to coloured nature of the test item but not any adverse signs.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
The noted non-toxicological and statistically significant increase in mean body weight on PND 42 in group G2 females when compared with vehicle control group .

A statistically significant decrease in mean body weights on PND 63, 70, 77, 84 and 91 and statistically significant decrease in percent change in mean body weight gain with respect to postnatal day (PND) 21 on PND 63, 70, 77, 84 and 91 in groups G3 and G4 males were noted when compared with vehicle control group.
These changes are not considered as adverse as there were no other systemic toxicity effects noted in any of these tested dose groups but are considered as stress induced effects due to test item administration.
Cohort 1B (C1B)
The noted non-toxicological and statistically significant increase in mean body weights on PND 77 and 84 in group G2-C1B females; increase in mean body weights on
PND 77, 84, 91 and 98 in group G3-C1B females; increase in mean body weights on PND 84, 91 and 98 in group G4-C1B females; increase in percent change in mean
body weight gain with respect to postnatal day (PND) 21 on PND 84 in group
G3-C1B females when compared with vehicle control group are considered as incidental.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
A statistically significant decrease in mean feed consumption during week 4 in group G3-C1A males when compared with vehicle control group is considered as incidental.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in mean haemoglobin, haematocrit, and prothrombin time levels in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in mean glucose, creatinine, cholesterol, and albumin levels of group G4 males; statistically significant decrease in mean creatinine levels in all the tested dose group females; statistically significant decrease in mean albumin levels in group G3 females was noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)

In group G2-C1A,
- decrease in mean absolute weight of liver and increase in relative weight of brain (males).
- increase in mean absolute and relative weight of liver and increase in absolute weight of iliac lymph nodes (females).

In group G3-C1A,
- decrease in mean terminal body weight (males).
- increase in mean relative weight of adrenals, kidneys and thyroid along with parathyroid (males).
- increase in mean relative weight of liver (females).

In group G4-C1A,
- decrease in mean terminal body weight (males).
- decrease in mean absolute weight of epididymides, heart, brain, liver (males); increase in mean relative weight of adrenals, testes, kidneys, prostate and thyroid along with parathyroid (males).
- increase in mean absolute and relative weight of thymus (females).

Cohort 1B (C1B)

In group G2-C1B,
- decrease in mean relative weight of mandibular lymph nodes (females).
In group G3-C1B,
- decrease in mean relative weight of adrenals and thyroid along with parathyroid (males).
- increase in mean absolute weight of spleen (females).
In group G4-C1B,
- increase in mean terminal body weight (females).
- decrease in mean absolute and relative weight of adrenals (males).
- decrease in mean relative weight of iliac and mandibular lymph nodes (females).

These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also neither gross pathological changes nor microscopic changes were noted in any of these organs in all the tested dose group animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In test-item administered group of animals of both the sexes, the contents of gastrointestinal tract were found to be pink in colour which could be attributed to physical appearance of the test item (pink in colour).
A single incidence of small sized testis was observed in G3-C1A group male rat and was microscopically correlated with moderate atrophy of tubules. This change was considered as an isolated spontaneous finding in rats.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few of the microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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.
Quantitative ovarian follicular assessment (primordial and primary follicles) in all parental and C1A females of control and high dose groups did not reveal any test item related variations. Similarly, quantitative evaluation of corpora luteal count in
C1A females of control and high dose group did not reveal any test item related variations.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Splenic Lymphocyte Sub-populations statistically significant increase in mean T cytotoxic (CD8+) cells populations in group G4-C1A males; increase in mean T cytotoxic (CD8+) cells populations in group G3-C1A females; decrease in T "helper" (CD4+) cells populations in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment as the obtained mean value is within in-house historical control range of same species and strain.

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day)
when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to
102 (for C1B animals) under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Generation:

other: cohort 1A and cohort 1B

Effect level:

>= 1 000

Based on:

test mat.

Remarks:

the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: Splenic Lymphocyte Subpopulation Analysis

Remarks on result:

other: the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods up to weaning of F1 animals (P females) as well as when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to 102 (for C1B animals) under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Executive summary:

The objective of this Extended One-Generation Reproductive Toxicity (EOGRT) Study in Sprague Dawley Rats was to evaluate the effects of test item as a result of pre and postnatal exposure on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females, young and adult offspring.
The study was conducted to serve as a test for reproductive endpoints that required the interaction of males with females, females with conceptus, females with offspring and the F1 generation before and after sexual maturity. This study was also conducted to evaluate the spermatogenesis for males and oestrous cycles, follicle counts/oocyte maturation and ovarian integrity (histopathology) for females.

This EOGRT study was also conducted to evaluate gonadal function, the oestrus cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition and lactation. Furthermore, the present EOGRT study was conducted to provide detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, and was expected to identify specific target organs in the offspring. In addition, this study provided and/or confirmed information about the effects of Hostaperm Pink E on the integrity and performance of the adult male and female reproductive systems.

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males
+ 80 females)]. Each cohort consisted of four groups and consisted of 20 males and
20 females per group. The animals in G1 (P generation) and G1-C1A/G1-C1B
(F1 generation) groups were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], the animals in G2 (P generation) and G2-C1A/G2-C1B (F1 generation),
G3 (P generation) and G3-C1A/G3-C1B (F1 generation), and G4 (P generation) and G4-C1A/G4-C1B (F1 generation) groups were administered with Hostaperm Pink E at the dose levels of 111, 333 and 1000 mg/kg body weight/day respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115 at the concentrations of 10.0 mg/mL and
100.0 mg/mL. Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1, 8, 16 and 26 of the treatment periods and the mean results were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

All the P generation animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) was recorded once weekly for all the P animals. The assessment for haematology, clinical chemistry, urinalysis, and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each P generation group at termination. Sperm parameters (motility, morphology, and sperm concentration/daily sperm production) were estimated for all P males. Oestrus cyclicity evaluation was determined during pre-mating and cohabitation period for all P females.
The body weights and feed consumption were recorded during gestation (Gestation Day 0, 7, 14 and 20) and during lactation (Lactation Day 1, 4, 7, 14 and 21) for all the
P females. The gross pathology and organ weighing was performed on the day of termination for all the P animals. Histopathological examination was conducted on all the tissues collected from the P generation vehicle control and high dose group animals. All the P males and females were evaluated for reproductive performance or indices such as, mating and fertility index (for P males and females) and pre-coital interval, gestation length, fecundity index, gestation index, post-implantation loss and postnatal loss (for females). All P females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter).

All the P males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups. However, in test-item administered group animals of both the sexes, the faecal matter was noted with ‘pink’ in colour which could be attributed to physical appearance of the test item (pink in colour). There were no test item-related histopathological findings noted in high dose parental animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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 of this strain and age.

The F1 generation pups were observed once daily for external examinations and
twice daily for mortalities till termination, weighed individually on postnatal day (PND) 1, 4, 7, 14 and 21, observed for occurrences of postnatal developmental landmarks, observed for responses towards to sensory reflexes during postnatal period, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13, observed for gross pathological observations at termination. The assessment for serum Thyroxine (T4) levels was conducted for PND 4 pups and assessment for serum Thyroxine (T4) and Thyroid Stimulating Hormone levels was conducted for PND 21 pups.

There were no test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels in F1 generation pups. No gross pathological changes noted in of the any of the F1 pups of both sexes from all the tested dose group litters.

All the Cohort 1A (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1A animals were evaluated for occurrence of sexual maturation (for balanopreputial separation in C1A males and for vaginal patency in C1A females). All C1A females were evaluated for mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells. All C1A females were evaluated for oestrus cyclicity from PND 75 to until sacrifice. The assessment for haematology, clinical chemistry, urinalysis and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each C1A group at termination. Sperm parameters (motility, morphology and daily sperm production) were estimated for all C1A males. The gross pathology and organ weighing were performed on the day of termination for all the C1A animals. Histopathological examination was conducted on all the tissues collected from the
C1A vehicle control and high dose group animals. The flow cytometric analysis of splenic samples for assessment of splenic lymphocyte sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes was conducted for randomly selected males and females from each C1A group.

All the C1A males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups when compared with vehicle control group, except slight reduction in mean body weight and percent change in mean body weight gain in all the tested dose group males. There were no changes noted in sub-populations of splenic lymphocytes such as, T "helper" (CD4+) cells,
T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes at any of the tested dose groups when compared with vehicle control group. There were no test item-related histopathological findings noted in high dose C1A animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus 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.

All the Cohort 1B (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1B animals were evaluated for occurrence of sexual maturation (for balano-preputial separation in C1B males and for vaginal patency in C1B females). The gross pathology and organ weighing were performed on the day of termination for all the C1B animals.

All the C1B males and females did not reveal any test item related changes in systemic and reproductive endpoints, except reduction in mean body weight and percent change in mean body weight gain in all the tested dose group of both sexes.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 June 2021 to 22 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

As per OECD Guideline for Testing of Chemicals, Number 422, adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Justification for study design:

- Basis for dose level selection: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item.
The doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups for the dose range finding study (BIO-DTX 056) in consultation with the sponsor.
There were no test item-related related changes noted in any of the systemic, reproduction and developmental parameters up to the dose level of 1000 mg/kg body weight/day in the Dose Range Finding Study (BIO-DTX 056).
Hence, the same doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively, for the present study in consultation with the sponsor.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 21 to 29ºC
- Stability under test conditions: The test item was stable for 6 hours and 48 hours at room temperature in 0.5% w/v Carboxymethyl cellulose with the concentrations of 5 mg/mL and 100 mg/mL.
- Solubility of the test substance: The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results.
Hence, 0.5% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations in consultation with sponsor.
0.5% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of 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: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: Males: 254.36 to 291.64 g; Females: 200.54 to 211.92 g
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
During acclimatization, two animals of same sex were housed.
i. Main Group Animals:
- Pre mating: Per cage two animals of same sex and group were housed.
- Cohabitation Period (mating): Per cage two animals (one male and one female) of same group were housed.
- Post-mating: After confirmation on 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 for main group females from gestation day 20 onwards.
ii. Recovery Group Animals:
- Maximum of two animals per cage per group of same sex were housed, tunnels were provided as enrichment for the animals which were housed individually.
- Diet (e.g. ad libitum): Altromin maintenance diet
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 to 22.9 oC
- Humidity (%): 48 to 63%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 24 June 2021 To: 16 September 2021

Route of administration:

oral: gavage

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): After confirmation on 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 for main group females from gestation day 20 onwards.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1 and 5 of the treatment. The results of dose concentration verifications for the prepared test item formulations and the mean results of analysis were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was less than 15%.

Duration of treatment / exposure:

The test item or vehicle were administered to the animals through the oral (gavage) route once daily.
The males were treated for two weeks pre-mating, during mating period and up to the day before sacrifice (total of completion of 31 days of administration).
The females were treated for a two-week pre-mating period (14-days), during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 50 to 62 days).
The females with positive mating signal, but no-evidence of pregnancy / littering were treated during two-week pre-mating period, during mating and further 24 days from the day of positive mating signal (group G1 - one female until experimental day 39; group G2 - one female until experimental day 52, one female until experimental day 46; G3 - two females until experimental day 49; group G4 - one female until experimental day 39).
The recovery group animals were treated until the first scheduled sacrifice of dams (total of 52 days of administration) and kept without treatment for further 18-days of observation period.
The test item/vehicle was administered by oral (gavage) route using a stainless-steel intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 10 mL/kg body weight/day with the concentration of 11.1, 33.3 and 100 mg/mL for low, mid, and high dose groups, respectively. Vehicle was administered to the vehicle control group at an equivolume of 10 mL/kg body weight/day.
The actual dose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation.
The prepared formulations were stirred using magnetic stirrer during administration to maintain homogeneity.

Frequency of treatment:

Once Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 13 to 14 weeks

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low Dose

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid Dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

Main Group: 24 (12 Males + 12 Females)
Recovery Group: 10 (5 Males + 5 Females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item.
Based on the above available information, the doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups for the dose range finding study (BIO-DTX 056) in consultation with the sponsor.
There were no test item-related related changes noted in any of the systemic, reproduction and developmental parameters up to the dose level of 1000 mg/kg body weight/day in the Dose Range Finding Study (BIO-DTX 056).
Hence, the same doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively, for the present study in consultation with the sponsor.

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the main and recovery groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study does not exceed ±20% (Males: -6.80 to 11.14%; Females: -12.58 to 8.29%) of the mean body weight of each sex. The grouping was done one day prior to initiation of treatment. Body weight of the animals were analyzed statistically for mean body weight to rule out statistical significant differences between groups within each sex.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for general clinical signs of toxicity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Day 1 before treatment and weekly thereafter during treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group males were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.
The main group females were weighed at receipt, on the first day of dosing, weekly thereafter during pre-mating and until confirmation of mating. All the pregnant animals were weighed on gestation days (GD) 0, 7, 14 and 20 and on lactation days (LD) 1, 4, 7, 13 and 14 (terminal body weight). The non-pregnant animals were weighed weekly once until termination and the terminal body weights were recorded before scheduled sacrifice.

The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Females with normal oestrus cyclicity only were selected for the treatment. Vaginal smears were monitored daily from beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. The status of oestrus cyclicity of females was determined on termination day.

Recovery group females were not evaluated for oestrus cyclicity

Sperm parameters (parental animals):

Parameters examined in all males: testis weight, epididymis weight.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1] 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.

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

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals were sacrificed after completion of 31 days of treatment. The males were necropsied in randomized manner.
- Maternal animals: All pregnant females were sacrificed on lactation day 14 and the non-pregnant females were sacrificed after 24 days from the last day of confirmation of mating.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [22] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- Dead pups and pups which were sacrificed on PND 4/13, examined for gross abnormalities with particular attention to the external reproductive genitals and the findings were recorded.

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

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of an appendix) was verified with the raw data. After verification, the data was subjected to various statistical analyses using SPSS software version 22.
All analysis and comparisons were evaluated at 95% with the level of confidence of P<0.05 respectively, indicated by the aforementioned tests and were designated by the superscripts throughout the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.
Note: Data of non-pregnant females was excluded for mean calculations and statistical analysis for reproductive end points but considered for systemic parameters. However, the individual animal data is presented in appendices.
The statistical analysis tests followed for the interpretation of data are as mentioned below:

Parametric - One-way ANOVA
• Body weight (weekly/gestation/lactation)
• Percent Change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Hematology
• Clinical chemistry
• Urinalysis
• Absolute/relative organ weights
• Functional Observation parameters
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum T4 values (adults/pups).

Non Parametric - Kruskal-Wallis
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter
• Litter size
• No. of early resorptions/litter
• No. of late resorptions/litter
• Prenatal loss/litter
• Postnatal loss/litter

Cross Tabs - Chi-square test
• Reproductive performances (frequency occurrences)
- mated/non-mated;
- fertile/infertile;
- pregnant/non-pregnant;
- with/without live pups;
- with/without dead pups

Reproductive indices:

The following reproductive performance indices were calculated as mentioned below and presented in the study report,

Male Mating Index (%) = No. of males with confirmed mating / Total No. of males cohabited x 100
Female Mating Index (%) = No. of sperm-positive females / Total No. of females cohabited x 100
Male Fertility Index (%) = No. of males impregnating a female / Total No. of males mated x 100
Female Fertility Index (%) = No. of pregnant females / No. of sperm-positive females x 100
Pre-coital Interval (Days) = Date of confirmation of mating – Date of initiation of cohabitation
Gestation Length (days) = Date of parturition – Date of positive evidence of mating (GD 0)
Gestation Index (%) = No. of females with live born / No. of females with evidence of pregnancy x 100
Parturition Index (%) = No. of females littered / No. of females with evidence of pregnancy x 100
Pregnancy Index (%) = No. of pregnant females / No. of females with confirmed mating x 100

Offspring viability indices:

Sex ratio (m/f) = No. of male offspring / Number of female offspring
Live Birth Index (%) per litter = No. of pups born alive / Total No. of pups born x 100
Pup Survival index (%) on LD 4/7/13 = Total No. of live pups on LD 4/7/13 / No. of pups born/4/7/13 x 100
Sex ratio (m/f) on LD 4/7/13 = No. of male offspring on LD 4/7/13 / No. of female offspring on LD 4/7/13
Post-Implantation Loss (%) = No. of implantations - No. of viable pups / No. of implantations x 100
Post-implantation Loss (No.) = No. of implantations - No. of live births
Postnatal loss on LD 13 (%) = No. of pups alive on postnatal day 13 /No. of live pups at birth x 100
Postnatal Loss on LD 13 (No.) = Live births - pups alive at lactation day 13
Anogenital Distance (AGD) Ratio = Anogenital distance (mm) / Cube root of PND 4 pup weight x 100

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

ophthalmological examination

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other: Thyroid hormonal levels

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes in mean serum T4 hormone levels of pups at postnatal day 13 in any of the tested dose group litters when compared with vehicle control group litters. However, an incidental statistically significant decrease in mean T4 levels in pups was noted in groups G2, G3 and G4 when compared with vehicle control group.

Description (incidence and severity):

The oral administration of test item Hostaperm Red Violet ER 02 over the time of the study, did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

gross pathology

other: Serum Thyroid hormonal levels (T4)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

TABLE 1.     SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	N (12)	NAD (12)	0 (12)
G2, M & 111	12	N (12)	NAD (12)	0 (12)
G3, M & 333	12	N (12)	NAD (12)	0 (12)
G4, M & 1000	12	N (12)	NAD (12)	0 (12)

M: Male; N: Normal; NAD: No Abnormality Detected.
a: observed once daily; b: observed once weekly; c: observed twice daily.

Note:Dark purple colored faeces were noted from day 2 observation throughout the experiment period till termination in group G3 & G4 animals.

TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, F & 0	12	N (12)	NAD (12)	0 (12)
G2, F & 111	12	N (12)	NAD (12)	0 (12)
G3, F & 333	12	N (12)	NAD (12)	0 (12)
G4, F & 1000	12	N (12)	NAD (12)	0 (12)

F: Female; N: Normal; NAD: No Abnormality Detected.
a: observed once daily; b: observed once weekly; c: observed twice daily.

Note:Dark purple colored faeces were noted from day 2 observation throughout the experiment period till termination in group G3 & G4 animals.

TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY, DETAILED CLINICAL EXAMINATION AND MORTALITY RECORD

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1R, M & 0	5	N (5)	NAD (5)	0 (5)
G4R, M & 1000	5	N (5)	NAD (5)	0 (5)
G1R, F & 0	5	N (5)	NAD (5)	0 (5)
G4R, F & 1000	5	N (5)	NAD (5)	0 (5)

M : Male; F: Female; R: Recovery; N: Normal; NAD: No abnormality detected.
a: observed once daily; b: observed once weekly; c: observed twice daily. Note:Dark purple colored faeces were noted from day 2 throughout the experiment period till termination in group G4R animals.                 

TABLE 2.     SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28
G1, M & 0	Mean	400.75	421.20	438.19	446.01	458.77
	±SD	22.07	32.34	36.49	37.08	38.53
	n	12	12	12	12	12
G2, M & 111	Mean	401.74	417.26	433.41	442.75	452.56
	±SD	20.39	24.35	30.70	31.29	30.16
	n	12	12	12	12	12
G3, M & 333	Mean	402.72	419.51	438.54	444.84	456.36
	±SD	19.35	23.54	30.11	27.23	24.95
	n	12	12	12	12	12
G4, M & 1000	Mean	401.99	422.76	443.74	453.94	463.38
	±SD	21.50	27.21	34.32	36.83	39.17
	n	12	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals.

TABLE 2 (Contd…). SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21^#
G1, F & 0	Mean	250.88	257.35	267.91	280.34
	±SD	14.86	17.17	18.25	26.26
	n	12	12	12	3
G2, F & 111	Mean	251.05	256.43	266.53	272.56
	±SD	12.78	15.62	15.30	25.98
	n	12	12	12	3
G3, F & 333	Mean	251.13	256.32	261.08	260.46
	±SD	13.59	14.00	15.94	17.45
	n	12	12	12	4
G4, F & 1000	Mean	251.24	261.46	265.51	269.05
	±SD	15.56	18.91	20.71	24.37
	n	12	12	12	4

F: Female; SD: Standard Deviation; n: Number of Animals.

#: The data obtained from females in cohabitation only considered for mean calculations.

The data of day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

TABLE 2 (Contd…). SUMMARY OF BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63
G1R, M & 0	Mean	395.45	407.17	422.63	436.11	448.75	458.35	473.23	489.33	502.87	514.37
	±SD	21.72	21.10	31.29	32.45	32.78	34.62	33.67	33.58	34.92	37.99
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	396.36	412.35	430.15	444.31	454.98	464.82	475.04	493.68	508.20	519.69
	±SD	20.86	20.17	12.53	16.96	17.27	14.72	15.50	9.28	11.65	11.69
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	245.24	250.97	262.44	268.72	277.56	284.92	295.96	306.72	315.86	324.75
	±SD	10.97	10.11	13.68	15.80	15.60	15.72	19.40	19.59	17.41	15.21
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	245.66	248.99	264.41	270.43	278.43	286.33	298.35	308.32	318.51	327.87
	±SD	12.82	12.43	13.84	12.16	11.19	12.10	12.79	13.72	10.97	12.44
	n	5	5	5	5	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 3.     SUMMARY RECORD OF GESTATION BODY WEIGHT (g)

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	7	14	20
G1, F & 0	Mean	272.42	287.38	319.92	375.62
	±SD	18.33	21.41	28.89	35.87
	n	11	11	11	11
G2, F & 111	Mean	269.81	286.87	328.45	391.71
	±SD	11.98	12.37	15.11	22.46
	n	10	10	10	10
G3, F & 333	Mean	264.91	282.42	317.51	369.85
	±SD	15.69	14.58	20.68	32.90
	n	10	10	10	10
G4, F & 1000	Mean	273.49	291.35	327.07	382.89
	±SD	17.37	17.37	23.26	39.30
	n	11	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.

TABLE 4.     SUMMARY RECORD OF LACTATION BODY WEIGHT (g)

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
		1	4	7	13
G1, F & 0	Mean	294.60	300.48	308.18	323.25
	±SD	22.07	22.28	22.06	20.39
	n	11	11	11	11
G2, F & 111	Mean	306.24	309.48	314.78	330.57
	±SD	22.46	21.39	20.72	19.26
	n	10	10	10	10
G3, F & 333	Mean	293.35	297.31	304.48	319.65
	±SD	19.47	20.23	20.46	22.22
	n	10	10	10	10
G4, F & 1000	Mean	303.31	306.82	313.01	325.64
	±SD	22.77	24.10	23.73	23.27
	n	11	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams.

TABLE 5.     SUMMARY OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 28
G1, M & 0	Mean	5.01	9.25	11.19	14.36
	±SD	3.10	4.96	4.86	4.98
	n	12	12	12	12
G2, M & 111	Mean	3.84	7.80	10.13	12.59
	±SD	2.04	3.09	3.48	3.20
	n	12	12	12	12
G3, M & 333	Mean	4.15	8.89	10.50	13.42
	±SD	2.15	5.14	5.14	5.61
	n	12	12	12	12
G4, M & 1000	Mean	5.12	10.29	12.82	15.16
	±SD	1.91	3.83	4.55	5.27
	n	12	12	12	12

M: Male; SD: Standard Deviation; n: Number of Animals.

TABLE 5 (Contd…). SUMMARY OF PERCENTCHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21^#
G1, F & 0	Mean	2.56	6.78	10.07
	±SD	2.21	3.23	3.01
	n	12	12	3
G2, F & 111	Mean	2.12	6.21	9.94
	±SD	2.46	4.16	1.49
	n	12	12	3
G3, F & 333	Mean	2.08	3.94	5.98
	±SD	1.81	2.08	1.64
	n	12	12	4
G4, F & 1000	Mean	4.05	5.61	9.37
	±SD	3.24	3.20	3.75
	n	12	12	4

F: Female; SD: Standard Deviation; n: Number of Animals.

#: The data obtained from females in cohabitation only considered for mean calculations. The data of day 1 to 21 body weight was not subjected to statistical analysis due to uneven number of variables.

TABLE 5 (Contd…). SUMMARY OF PERCENTCHANGE IN BODY WEIGHT GAIN (%) WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 35	1 to 42	1 to 49	1 to 56	1 to 63
G1R, M & 0	Mean	2.98	6.83	10.25	13.47	15.92	19.69	23.73	27.13	30.00
	±SD	0.92	4.09	4.67	5.29	6.27	5.81	4.88	4.25	4.30
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	4.06	8.66	12.20	14.91	17.44	20.09	24.77	28.45	31.32
	±SD	1.73	3.60	3.33	3.84	4.90	6.78	5.51	6.10	5.14
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	2.35	7.00	9.55	13.20	16.19	20.62	25.02	28.77	32.44
	±SD	0.72	2.36	3.15	4.61	4.45	4.14	4.54	3.55	3.44
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	1.37	7.66	10.20	13.45	16.65	21.52	25.60	29.78	33.57
	±SD	1.07	2.71	4.80	4.07	3.73	3.02	4.10	3.75	3.31
	n	5	5	5	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 6.     SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING GESTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	5.46	11.22	17.44
	±SD	1.77	2.82	4.18
	n	11	11	11
G2, F & 111	Mean	6.35	14.52	19.23
	±SD	2.12	3.20	3.16
	n	10	10	10
G3, F & 333	Mean	6.67	12.40	16.35
	±SD	1.92	3.55	3.43
	n	10	10	10
G4, F & 1000	Mean	6.56	12.23	16.88
	±SD	1.60	3.35	4.81
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis, but presented in individual animal data.

TABLE 7.     SUMMARY RECORD OF PERCENT CHANGE IN BODY WEIGHT GAIN (%) DURING LACTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	2.01	2.58	4.96
	±SD	0.96	0.73	1.51
	n	11	11	11
G2, F & 111	Mean	1.08	1.74	5.07
	±SD	0.86	0.95	1.60
	n	10	10	10
G3, F & 333	Mean	1.35	2.42	4.98
	±SD	0.95	1.29	1.32
	n	10	10	10
G4, F & 1000	Mean	1.14	2.04	4.06
	±SD	1.00	0.93	0.83
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams.

TABLE 8.     SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
		Week 1	Week 2
G1, M & 0	Mean	21.31	24.01
	±SD	2.38	1.81
	n	12 (6)	12 (6)
G2, M & 111	Mean	21.39	23.12
	±SD	1.76	0.75
	n	12 (6)	12 (6)
G3, M & 333	Mean	22.25	23.93
	±SD	2.39	1.85
	n	12 (6)	12 (6)
G4, M & 1000	Mean	22.28	24.17
	±SD	1.23	0.67
	n	12 (6)	12 (6)

M: Male; SD: Standard Deviation; n: Number of Animals (Number of cages).

TABLE 8 (Contd…). SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Pre-mating Period
		Week 1	Week 2
G1, F & 0	Mean	18.14	19.17
	±SD	1.56	1.22
	n	12 (6)	12 (6)
G2, F & 111	Mean	17.79	20.11
	±SD	1.85	1.70
	n	12 (6)	12 (6)
G3, F & 333	Mean	18.26	19.22
	±SD	1.99	1.31
	n	12 (6)	12 (6)
G4, F & 1000	Mean	18.43	20.07
	±SD	2.28	1.22
	n	12 (6)	12 (6)

F: Female; SD: Standard Deviation; n: Number of Animals (Number of cages).

TABLE 8 (Contd…). SUMMARY OF FEED CONSUMPTION (g/animal/day) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day)
		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9
G1R, M & 0	Mean	23.52	24.34	25.11	26.35	27.86	29.05	30.75	31.83	32.47
	±SD	3.71	1.67	1.55	1.27	0.97	1.11	0.47	3.66	3.01
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G4R, M & 1000	Mean	22.84	23.75	24.26	27.17	28.86	30.05	31.07	32.19	33.03
	±SD	0.77	1.04	1.34	2.09	2.61	2.18	2.12	1.59	1.67
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G1R, F & 0	Mean	17.44	18.36	19.45	10.21	23.17	24.20	25.35	26.19	27.04
	±SD	1.79	0.40	0.40	9.72	0.99	0.82	0.62	0.41	0.99
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)
G4R, F & 1000	Mean	17.21	19.05	20.42	11.29	24.18	25.37	26.19	27.14	27.89
	±SD	0.69	1.77	0.91	10.38	0.03	0.80	0.84	0.69	0.68
	n	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)	5 (3)

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals(Number of cages).

TABLE 9.     SUMMARY RECORD OF FEED CONSUMPTION (g/animal/day) DURING GESTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	19.02	21.07	25.81
	SD	0.70	1.95	1.63
	n	11	11	11
G2, F & 111	Mean	19.23	22.20	26.84
	SD	0.88	1.81	1.78
	n	10	10	10
G3, F & 333	Mean	19.00	21.49	26.11
	SD	0.74	1.39	1.26
	n	10	10	10
G4, F & 1000	Mean	19.43	22.01	26.67
	SD	0.58	1.70	1.97
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams.

Note: Excluded the data of non-pregnant females for mean calculations and statistical analysis but presented in individual animal data.

TABLE 10.     SUMMARY RECORD OF FEED CONSUMTION (g/animal/day) DURING LACTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		Feed Consumption (g/animal/day) during Lactation Day (LD)
		1 to 4	4 to 7	7 to 13
G1, F & 0	Mean	25.21	35.80	40.88
	SD	1.20	7.26	1.43
	n	11	11	11
G2, F & 111	Mean	24.58	32.72	41.09
	SD	1.34	0.99	0.98
	n	10	10	10
G3, F & 333	Mean	25.11	32.75	40.54
	SD	1.42	1.16	0.94
	n	10	10	10
G4, F & 1000	Mean	23.94	33.14	39.84
	SD	5.99	1.28	1.03
	n	11	11	11

F: Female; SD: Standard Deviation; n: Number of Dams.

TABLE 11.     SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)
Group, Sex & Dose (mg/kg body weight/day) →	G1, M & 0		G2, M & 111		G3, M & 333		G4, M & 1000
Number of Animals→	12		12		12		12
Observations↓
Eye→ Region↓	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N

M: Male; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)
Group, Sex & Dose (mg/kg body      weight/day) →	G1, F & 0		G2, F & 111		G3, F & 333		G4, F & 1000
Number of Animals→	12		12		12		12
Observations↓
Eye→ Region↓	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N

F: Female; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)
Group, Sex & Dose (mg/kg body      weight/day) →	G1R, M & 0		G4R, M & 1000
Number of Animals→	5		5
Observations↓
Eye→ Region↓	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N

M: Male; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day -1 (Grouping and randomization)
Group, Sex & Dose (mg/kg body      weight/day) →	G1R, F & 0		G4R, F & 1000
Number of Animals→	5		5
Observations↓
Eye→ Region↓	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N

F: Female; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day 30
Group, Sex & Dose (mg/kg body weight/day) →	G1, M & 0		G2, M & 111		G3, M & 333		G4, M & 1000
Number of Animals→	12		12		12		12
Observations↓
Eye→ Region↓	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
Other findings:	N	N	N	N	N	N	N	N

M: Male; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Pregnant/Littered females: Lactation day 13; Non-pregnant females: (Experimental day 39 to 52)
Group, Sex & Dose (mg/kg body weight/day) →	G1, F & 0		G2, F & 111		G3, F & 333		G4, F & 1000
Number of Animals→	12		12		12		12
Observations↓
Eye→ Region↓	LE	RE	LE	RE	LE	RE	LE	RE
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
Other findings:	N	N	N	N	N	N	N	N

F: Female; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day 65
Group, Sex & Dose (mg/kg body weight/day) →	G1R, M & 0		G4R, M & 1000
Number of Animals→	5		5
Observations↓
Eye→ Region↓	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N
Other findings:	N	N	N	N

M: Male; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 11 (Contd…). SUMMARY OF OPHTHALMOLOGICAL EXAMINATION RECORD

Day 65
Group, Sex & Dose (mg/kg body weight/day) →	G1R, F & 0		G4R, F & 1000
Number of Animals→	5		5
Observations↓
Eye→ Region↓	LE	RE	LE	RE
Eye Lids	N	N	N	N
Cornea	N	N	N	N
Iris	N	N	N	N
Aqueous Humour	N	N	N	N
Lens	N	N	N	N
Vitreous Humour	N	N	N	N
Retina/Optic disc	N	N	N	N
Other findings:	N	N	N	N

F: Female; R: Recovery; N: Normal; LE: Left Eye; RE: Right Eye.

TABLE 12.     SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Day 30
Parameters↓	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M
	Dose (mg/kg body weight/day)		0	111	333	1000
	Number of Animals (randomly selected)		5	5	5	5
Home Cage Observations
Home cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral closure			1	1	1	1
Handling Observations
Ease of removal from the cage			2	2	2	2
Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur appearance			1	1	1	1
Piloerection			1	1	1	1
Eye prominence			1	1	1	1
Muscle tone			1	1	1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Day 30
Parameters↓	Group & Sex		G1 & M	G2 & M	G3 & M	G4 & M
	Dose (mg/kg body weight/day)		0	111	333	1000
	Number of Animals (randomly selected)		5	5	5	5
Open Field Observations
Mobility			1	1	1	1
Gait			1	1	1	1
Arousal			3	3	3	3
Number of rearing		Mean	4.4	4.8	4.8	4.8
		±SD	1.1	0.8	0.8	0.8
Number of urine pools		Mean	3.4	3.8	3.8	3.8
		±SD	0.5	0.8	0.8	0.8
Number of defecations		Mean	3.4	3.2	3.4	3.2
		±SD	0.5	0.8	0.5	0.4
Clonic involuntary movement			1	1	1	1
Tonic involuntary movement			1	1	1	1
Stereotype behaviour			1	1	1	1
Number of grooming		Mean	3.4	3.2	3.4	3.2
		±SD	0.5	0.8	0.5	0.8
Sensory Observations
Approach response			2	2	2	2
Auditory response			2	2	2	2
Touch response			2	2	2	2
Pupil reflex			2	2	2	2
Tail pinch response			2	2	2	2
Righting reflex			1	1	1	1
Physiological Observation
Body temperature (°F)		Mean	98.4	98.4	98.1	98.4
		±SD	0.5	0.4	0.3	0.5
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Lactation Day 13
Parameters↓	Group & Sex		G1 & F	G2 & F	G3 & F	G4 & F
	Dose (mg/kg body weight/day)		0	111	333	1000
	Number of Animals (randomly selected)		5	5	5	5
Home Cage Observations
Home cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral closure			1	1	1	1
Handling Observations
Ease of removal from the cage			2	2	2	2
Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur appearance			1	1	1	1
Piloerection			1	1	1	1
Eye prominence			1	1	1	1
Muscle tone			1	1	1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Lactation Day 13
Parameters↓	Group & Sex		G1 & F	G2 & F	G3 & F	G4 & F
	Dose (mg/kg body weight/day)		0	111	333	1000
	Number of Animals (randomly selected)		5	5	5	5
Open Field Observations
Mobility			1	1	1	1
Gait			1	1	1	1
Arousal			3	3	3	3
Number of rearing		Mean	4.8	4.8	4.8	4.8
		±SD	0.8	0.8	0.8	0.8
Numbers of urine pools		Mean	3.6	3.8	3.6	3.8
		±SD	0.5	0.8	0.5	0.8
Number of defecations		Mean	3.4	3.0	2.8	3.2
		±SD	0.9	0.7	0.8	0.4
Clonic involuntary movement			1	1	1	1
Tonic involuntary movement			1	1	1	1
Stereotype behaviour			1	1	1	1
Number of grooming		Mean	3.2	3.2	2.6	3.0
		±SD	0.4	0.8	0.5	0.7
Sensory Observations
Approach response			2	2	2	2
Auditory response			2	2	2	2
Touch response			2	2	2	2
Pupil reflex			2	2	2	2
Tail pinch response			2	2	2	2
Righting reflex			1	1	1	1
Physiological Observation
Body temperature (°F)		Mean	98.4	98.4	98.8	98.7
		±SD	0.6	0.5	0.5	0.8
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65
Parameters↓	Group & Sex		G1R & M	G4R & M
	Dose (mg/kg body weight/day)		0	1000
	Number of Animals		5	5
Home Cage Observations
Home cage posture			1	1
Respiratory pattern			1	1
Clonic involuntary movements			1	1
Tonic involuntary movements			1	1
Vocalization			1	1
Palpebral closure			1	1
Handling Observations
Ease of removal from the cage			2	2
Ease of handling animal in hand			2	2
Red or crusty deposits		Eyes	1	1
		Nose	1	1
		Mouth	1	1
Lacrimation			1	1
Salivation			1	1
Fur appearance			1	1
Piloerection			1	1
Eye prominence			1	1
Muscle tone			1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65
Parameters↓	Group & Sex		G1R & M	G4R & M
	Dose (mg/kg body weight/day)		0	1000
	Number of Animals		5	5
Open Field Observations
Mobility			1	1
Gait			1	1
Arousal			3	3
Number of rearing		Mean	5.0	4.4
		±SD	1.0	1.1
Numbers of urine pools		Mean	3.6	3.4
		±SD	0.5	0.5
Number of defecations		Mean	3.6	3.4
		±SD	0.5	0.5
Clonic involuntary movement			1	1
Tonic involuntary movement			1	1
Stereotype behaviour			1	1
Number of grooming		Mean	3.6	3.2
		±SD	0.5	0.4
Sensory Observations
Approach response			2	2
Auditory response			2	2
Touch response			2	2
Pupil reflex			2	2
Tail pinch response			2	2
Righting reflex			1	1
Physiological Observation
Body temperature (°F)		Mean	98.5	98.6
		±SD	0.5	0.5
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65
Parameters↓	Group & Sex		G1R & F	G4R & F
	Dose (mg/kg body weight/day)		0	1000
	Number of Animals		5	5
Home Cage Observations
Home cage posture			1	1
Respiratory pattern			1	1
Clonic involuntary movements			1	1
Tonic involuntary movements			1	1
Vocalization			1	1
Palpebral closure			1	1
Handling Observations
Ease of removal from the cage			2	2
Ease of handling animal in hand			2	2
Red or crusty deposits		Eyes	1	1
		Nose	1	1
		Mouth	1	1
Lacrimation			1	1
Salivation			1	1
Fur appearance			1	1
Piloerection			1	1
Eye prominence			1	1
Muscle tone			1	1
Home Cage Observations:Home cage posture - 1=Normal; Respiratory pattern - 1=Normal; Clonic involuntary movements - 1=None; Tonic involuntary movements - 1=None; Vocalization - 1=None; Palpebral closure - 1=Normal;Handling Observations:Ease of removal from the cage - 2=Normal; Ease of handling animal in hand - 2=Normal; Red or crusty deposits - 1=Absent; Lacrimation - 1=None; Salivation- 1=None; Fur appearance - 1=Normal; Piloerection - 1=None; Eye Prominence - 1=Normal; Muscle tone - 1=Normal

TABLE 12 (Contd…). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Day 65
Parameters↓	Group & Sex		G1R & F	G4R & F
	Dose (mg/kg body weight/day)		0	1000
	Number of Animals		5	5
Open Field Observations
Mobility			1	1
Gait			1	1
Arousal			3	3
Number of rearing		Mean	5.0	4.8
		±SD	1.0	0.8
Numbers of urine pools		Mean	3.6	3.8
		±SD	0.5	0.8
Number of defecations		Mean	3.4	3.0
		±SD	0.9	0.7
Clonic involuntary movement			1	1
Tonic involuntary movement			1	1
Stereotype behaviour			1	1
Number of grooming		Mean	3.2	3.2
		±SD	0.4	0.8
Sensory Observations
Approach response			2	2
Auditory response			2	2
Touch response			2	2
Pupil reflex			2	2
Tail pinch response			2	2
Righting reflex			1	1
Physiological Observation
Body temperature (°F)		Mean	98.4	98.3
		±SD	0.5	0.7
Open Field Observations: Mobility- 1=Normal;Gait- 1=Normal;Arousal- 3=Normal;Clonic involuntary movement- 1=None;Tonic involuntary movement -1=None;Stereotype behaviour -1=Absent;Sensory Observations:Approach response- 2=Normal;Auditory response- 2=Normal;Touch response -2=Normal;Pupil reflex- 2=Normal;Tail pinch response- 2=Normal;Righting reflex- 1=Present

TABLE 13.     SUMMARY OF LOCOMOTOR ACTIVITY / MOVEMENT COUNTS RECORD

Day 30
Group, Sex & Dose (mg/kg body weight/day)		Movement Counts (no.)
G1, M & 0	Mean	1911.4
	±SD	108.9
	n	5
G2, M & 111	Mean	1961.8
	±SD	138.9
	n	5
G3, M & 333	Mean	1954.2
	±SD	126.1
	n	5
G4, M & 1000	Mean	1931.4
	±SD	104.0
	n	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 13 (Contd…). SUMMARY OF LOCOMOTOR ACTIVITY / MOVEMENT COUNTS RECORD

Lactation Day 13
Group, Sex & Dose (mg/kg body weight/day)		Movement Counts (no.)
G1, F & 0	Mean	1923.4
	±SD	102.7
	n	5
G2, F & 111	Mean	1941.4
	±SD	125.1
	n	5
G3, F & 333	Mean	1989.0
	±SD	103.1
	n	5
G4, F & 1000	Mean	1973.2
	±SD	84.7
	n	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 13 (Contd…). SUMMARY OF LOCOMOTOR ACTIVITY / MOVEMENT COUNTS RECORD

Day 65
Group, Sex & Dose (mg/kg body weight/day)		Movement Counts (no.)
G1R, M & 0	Mean	2014.2
	±SD	210.7
	n	5
G4R, M & 1000	Mean	1980.6
	±SD	197.8
	n	5
G1R, F & 0	Mean	1943.4
	±SD	87.8
	n	5
G4R, F & 1000	Mean	1956.2
	±SD	91.6
	n	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 14.     SUMMARY OF AVERAGE GRIP STRENGTH (kgf) MEASUREMENT RECORD

Day 30
Group, Sex & Dose (mg/kg body weight/day)		Grip Strength (kgf)
		Forelimb	Hindlimb
G1, M & 0	Mean	1.529	0.510
	±SD	0.023	0.025
	n	5	5
G2, M & 111	Mean	1.501	0.539
	±SD	0.020	0.021
	n	5	5
G3, M & 333	Mean	1.521	0.552*
	±SD	0.048	0.008
	n	5	5
G4, M & 1000	Mean	1.539	0.544*
	±SD	0.040	0.015
	n	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 14 (Contd…). SUMMARY OF AVERAGE GRIP STRENGTH (kgf) MEASUREMENT RECORD

Lactation Day 13
Group, Sex & Dose (mg/kg body weight/day)		Grip Strength (kgf)
		Forelimb	Hindlimb
G1, F & 0	Mean	1.496	0.508
	±SD	0.009	0.008
	n	5	5
G2, F & 111	Mean	1.493	0.488
	±SD	0.010	0.015
	n	5	5
G3, F & 333	Mean	1.496	0.493
	±SD	0.009	0.021
	n	5	5
G4, F & 1000	Mean	1.498	0.499
	±SD	0.011	0.020
	n	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 14 (Contd…). SUMMARY OF AVERAGE GRIP STRENGTH (kgf) MEASUREMENT RECORD

Day 65
Group, Sex & Dose (mg/kg body weight/day)		Grip Strength (kgf)
		Forelimb	Hindlimb
G1R, M & 0	Mean	1.515	0.534
	±SD	0.020	0.035
	n	5	5
G4R, M & 1000	Mean	1.528	0.533
	±SD	0.029	0.009
	n	5	5
G1R, F & 0	Mean	1.516	0.499
	±SD	0.031	0.021
	n	5	5
G4R, F & 1000	Mean	1.503	0.481
	±SD	0.012	0.012
	n	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 15.     SUMMARY OF AVERAGE HINDLIMB FOOT SPLAY (cm) RECORD

Day 30
Group, Sex & Dose (mg/kg body weight/day)		Hindlimb Foot Splay (cm)
G1, M & 0	Mean	9.0
	±SD	1.0
	n	5
G2, M & 111	Mean	8.8
	±SD	1.3
	n	5
G3, M & 333	Mean	9.3
	±SD	0.6
	n	5
G4, M & 1000	Mean	8.6
	±SD	0.6
	n	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 15 (Contd…). SUMMARY OF AVERAGE HINDLIMB FOOT SPLAY (cm) RECORD

Lactation Day 13
Group, Sex & Dose (mg/kg body weight/day)		Hindlimb Foot Splay (cm)
G1, F & 0	Mean	8.0
	±SD	0.5
	n	5
G2, F & 111	Mean	7.9
	±SD	0.5
	n	5
G3, F & 333	Mean	8.1
	±SD	1.0
	n	5
G4, F & 1000	Mean	8.5
	±SD	0.9
	n	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 15 (Contd…). SUMMARY OF AVERAGE HINDLIMB FOOT SPLAY (cm) RECORD

Day 65
Group, Sex & Dose (mg/kg body weight/day)		Hindlimb Foot Splay (cm)
G1R, M & 0	Mean	8.6
	±SD	0.3
	n	5
G4R, M & 1000	Mean	9.0
	±SD	0.4
	n	5
G1R, F & 0	Mean	8.4
	±SD	0.4
	n	5
G4R, F & 1000	Mean	7.6*
	±SD	0.2
	n	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 16.     SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin	Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)	(MPV)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/µL)	(fL)
G1, M & 0	Mean	8.26	9.64	17.56	52.80	55.00	18.30	33.26	776.40	7.72
	±SD	1.95	1.09	1.34	4.49	3.09	1.14	0.53	330.86	0.52
	n	5	5	5	5	5	5	5	5	5
G2, M & 111	Mean	9.68	9.37	16.82	51.32	54.72	17.94	32.80	1135.20	7.46
	±SD	2.59	0.15	0.33	0.89	1.12	0.29	0.58	184.30	0.19
	n	5	5	5	5	5	5	5	5	5
G3, M & 333	Mean	8.67	9.40	16.58	50.78	54.12	17.64	32.62	962.60	7.72
	±SD	3.67	0.62	0.87	1.91	1.72	0.59	0.70	256.00	0.52
	n	5	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	8.14	9.35	16.88	52.02	55.74	18.06	32.46	1017.60	7.50
	±SD	2.42	0.54	0.55	1.58	2.71	0.74	0.53	93.02	0.38
	n	5	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	2.25	23.48	72.02	2.38	0.44	0.80
	±SD	1.24	9.09	9.80	1.15	0.17	0.49
	n	5	5	5	5	5	5
G2, M & 111	Mean	1.87	19.66	75.74	2.28	1.04	0.54
	±SD	0.64	3.44	3.55	0.74	0.59	0.23
	n	5	5	5	5	5	5
G3, M & 333	Mean	1.87	21.98	70.58	2.40	1.04	0.62
	±SD	0.60	5.35	7.57	2.37	0.34	0.61
	n	5	5	5	5	5	5
G4, M & 1000	Mean	2.01	20.86	75.78	1.42	0.72	0.54
	±SD	0.79	3.34	2.92	0.23	0.25	0.15
	n	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9cells/L)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	212.96	1.83	6.07	0.19	0.04	0.07	15.54	22.26
	±SD	101.87	0.42	2.01	0.08	0.02	0.06	0.78	6.12
	n	5	5	5	5	5	5	5	5
G2, M & 111	Mean	174.50	1.91	7.33	0.23	0.10*	0.05	15.72	25.28
	±SD	56.93	0.64	1.96	0.10	0.04	0.02	1.03	1.56
	n	5	5	5	5	5	5	5	5
G3, M & 333	Mean	175.20	1.80	5.98	0.27	0.09*	0.07	16.72	24.30
	±SD	59.25	0.52	1.97	0.39	0.03	0.11	1.49	1.59
	n	5	5	5	5	5	5	5	5
G4, M & 1000	Mean	184.78	1.70	6.17	0.11	0.06	0.04	15.98	26.78
	±SD	67.09	0.55	1.89	0.04	0.02	0.02	0.89	1.29
	n	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin	Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)	(MPV)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/µL)	(fL)
G1, F & 0	Mean	10.56	7.85	15.74	46.26	59.02	20.04	34.04	752.60	6.02
	±SD	3.78	0.45	0.65	3.16	4.16	1.08	0.91	361.58	0.64
	n	5	5	5	5	5	5	5	5	5
G2, F & 111	Mean	11.38	7.41	14.66	44.60	60.26	19.74	32.82	1130.00	6.72
	±SD	3.45	0.68	1.58	3.35	1.37	0.42	1.34	672.21	0.54
	n	5	5	5	5	5	5	5	5	5
G3, F & 333	Mean	9.62	7.95	15.56	46.34	58.34	19.60	33.60	1030.20	6.42
	±SD	3.95	0.28	0.72	2.05	3.12	0.81	1.07	104.22	0.57
	n	5	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	12.38	7.39	14.68	43.46	59.06	19.98	33.80	1003.00	6.92
	±SD	3.58	0.76	0.89	2.75	3.76	1.58	0.63	227.17	0.68
	n	5	5	5	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1, F & 0	Mean	2.28	32.72	60.66	3.68	1.46	0.60
	±SD	0.87	15.77	16.88	1.06	0.98	0.23
	n	5	5	5	5	5	5
G2, F & 111	Mean	4.52	30.00	62.56	4.60	1.18	0.42
	±SD	4.56	11.00	11.24	1.35	0.63	0.19
	n	5	5	5	5	5	5
G3, F & 333	Mean	1.49	25.26	66.92	4.46	1.22	0.80
	±SD	0.67	9.73	12.89	4.23	0.33	1.12
	n	5	5	5	5	5	5
G4, F & 1000	Mean	4.16	37.00	57.76	3.10	0.88	0.28
	±SD	2.72	20.56	20.80	0.97	0.47	0.11
	n	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9cells/L)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	177.36	3.64	6.21	0.40	0.15	0.06	17.60	23.34
	±SD	60.94	2.11	2.30	0.19	0.09	0.03	1.02	2.05
	n	5	5	5	5	5	5	5	5
G2, F & 111	Mean	313.86	3.59	6.91	0.56	0.12	0.05	22.06	27.48
	±SD	276.22	2.17	1.72	0.29	0.04	0.02	8.41	10.47
	n	5	5	5	5	5	5	5	5
G3, F & 333	Mean	117.60	2.64	6.29	0.39	0.12	0.06	16.88	22.08
	±SD	50.69	2.06	2.26	0.31	0.07	0.08	3.84	7.81
	n	5	5	5	5	5	5	5	5
G4, F & 1000	Mean	291.70	4.81	6.94	0.38	0.10	0.04	15.66	19.92
	±SD	145.98	3.66	3.06	0.17	0.03	0.02	3.85	5.41
	n	5	5	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin	Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)	(MPV)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/µL)	(fL)
G1R, M & 0	Mean	9.13	9.21	16.04	48.76	52.94	17.42	32.92	894.80	6.50
	±SD	0.78	0.14	0.43	1.79	1.78	0.39	0.42	134.10	0.31
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	9.55	9.14	15.80	48.54	53.08	17.30	32.62	1071.80	6.42
	±SD	1.05	0.32	0.65	2.25	0.91	0.30	0.57	268.87	0.37
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	6.97	7.65	14.26	42.16	55.20	18.64	33.78	873.60	6.32
	±SD	2.14	0.43	0.65	2.05	1.99	0.70	0.29	80.79	0.19
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	7.59	7.73	14.32	42.78	55.34	18.48	33.46	739.60	6.52
	±SD	3.51	0.22	0.57	1.63	2.15	0.70	0.25	253.21	0.13
	n	5	5	5	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1R, M & 0	Mean	1.63	25.28	69.00	1.76	2.30	0.88
	±SD	0.26	6.62	7.82	0.49	0.89	0.26
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	2.03	21.70	73.32	1.48	2.02	0.60
	±SD	0.50	5.52	5.80	0.37	0.36	0.29
	n	5	5	5	5	5	5
G1R, F & 0	Mean	1.84	26.12	68.68	2.30	1.34	0.68
	±SD	0.36	9.06	8.57	1.19	0.45	0.15
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	2.48*	29.02	64.16	2.40	2.86*	0.64
	±SD	0.39	7.89	6.96	0.87	1.30	0.13
	n	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 16 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute  Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9cells/L)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(Seconds)	(Seconds)
G1R, M & 0	Mean	150.40	2.27	6.35	0.16	0.21	0.08	16.00	21.56
	±SD	23.36	0.40	1.23	0.04	0.07	0.02	2.04	2.05
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	186.92	2.06	7.02	0.14	0.19	0.05	15.50	22.48
	±SD	52.68	0.53	1.09	0.04	0.04	0.03	1.17	3.12
	n	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	140.08	1.78	4.83	0.15	0.09	0.05	14.98	24.56
	±SD	23.05	0.73	1.73	0.05	0.02	0.01	0.53	1.98
	n	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	192.10*	2.06	4.98	0.17	0.25	0.05	15.92	19.34*
	±SD	30.17	0.60	2.77	0.07	0.24	0.02	1.17	2.34
	n	5	5	5	5	5	5	5	5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17.     SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin
		(GLU)	-	(CRE)	(CHO)	(TRI)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, M & 0	Mean	126.60	30.52	0.58	51.60	25.60	7.74	3.47
	±SD	15.47	2.56	0.03	3.36	2.30	0.26	0.19
	n	5	5	5	5	5	5	5
G2, M & 111	Mean	123.60	33.30	0.57	60.60	30.40	7.24*	3.55
	±SD	5.55	3.91	0.04	4.83	6.07	0.33	0.15
	n	5	5	5	5	5	5	5
G3, M & 333	Mean	122.60	33.78	0.60	58.00	29.40	7.44	3.65
	±SD	12.92	2.83	0.03	6.71	7.73	0.22	0.29
	n	5	5	5	5	5	5	5
G4, M & 1000	Mean	131.00	36.04	0.59	56.00	27.00	7.58	3.68
	±SD	19.79	4.26	0.02	9.57	6.16	0.29	0.24
	n	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1, M & 0	Mean	61.20	111.20	125.80	0.05	9.36	6.48
	±SD	16.93	12.34	21.11	0.01	0.11	0.58
	n	5	5	5	5	5	5
G2, M & 111	Mean	47.40	93.20	120.00	0.06	9.64	6.54
	±SD	8.96	13.46	31.92	0.03	0.47	0.62
	n	5	5	5	5	5	5
G3, M & 333	Mean	40.00	81.60*	127.20	0.06	10.40*	6.82
	±SD	5.96	12.30	38.01	0.03	1.05	0.62
	n	5	5	5	5	5	5
G4, M & 1000	Mean	52.20	91.80	114.80	0.06	9.94	6.62
	±SD	24.52	18.69	28.70	0.02	0.30	0.49
	n	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected) ; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Globulin	Albumin/ Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	-	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	4.27	0.82	14.24	141.64	3.96	104.10
	±SD	0.30	0.09	1.19	0.94	0.25	2.51
	n	5	5	5	5	5	5
G2, M & 111	Mean	3.69*	0.97	15.54	142.16	3.71	105.38
	±SD	0.38	0.12	1.83	0.53	0.15	1.40
	n	5	5	5	5	5	5
G3, M & 333	Mean	3.79*	0.97	15.77	142.44	3.86	105.78
	±SD	0.12	0.10	1.32	0.93	0.36	1.73
	n	5	5	5	5	5	5
G4, M & 1000	Mean	3.90	0.95	16.82	142.76	3.85	106.40
	±SD	0.29	0.11	1.99	0.66	0.09	0.86
	n	5	5	5	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals (randomly selected); *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin
		(GLU)	-	(CRE)	(CHO)	(TRI)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1, F & 0	Mean	102.20	48.68	0.54	76.60	78.60	7.32	3.63
	±SD	26.22	8.11	0.05	22.67	26.83	1.08	0.67
	n	5	5	5	5	5	5	5
G2, F & 111	Mean	121.20	51.14	0.52	67.80	59.40	7.34	3.41
	±SD	29.64	13.28	0.03	17.67	23.65	0.75	0.30
	n	5	5	5	5	5	5	5
G3, F & 333	Mean	103.80	44.78	0.57	75.60	43.20	7.56	3.58
	±SD	13.81	19.81	0.04	14.91	21.48	0.29	0.32
	n	5	5	5	5	5	5	5
G4, F & 1000	Mean	114.60	51.66	0.57	75.80	105.20	7.76	3.70
	±SD	16.44	16.16	0.07	20.44	68.28	0.58	0.11
	n	5	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1, F & 0	Mean	84.60	119.40	164.40	0.03	10.06	5.92
	±SD	14.43	14.31	75.62	0.04	1.06	0.95
	n	5	5	5	5	5	5
G2, F & 111	Mean	113.80	143.80	420.00	0.02	9.42	5.50
	±SD	66.08	71.78	367.29	0.02	0.64	1.64
	n	5	5	5	5	5	5
G3, F & 333	Mean	96.20	99.40	305.80	0.06	10.04	6.06
	±SD	82.35	16.01	412.83	0.02	0.85	1.93
	n	5	5	5	5	5	5
G4, F & 1000	Mean	88.60	101.80	212.40	0.03	10.38	6.38
	±SD	46.69	22.65	81.17	0.04	0.91	3.11
	n	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Globulin	Albumin/Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	-	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	3.69	0.98	22.72	143.04	4.02	106.18
	±SD	0.50	0.11	3.79	1.34	0.81	2.96
	n	5	5	5	5	5	5
G2, F & 111	Mean	3.93	0.88	23.87	142.18	3.57	107.22
	±SD	0.66	0.14	6.20	1.47	0.28	3.01
	n	5	5	5	5	5	5
G3, F & 333	Mean	3.98	0.91	20.90	142.36	3.60	107.94
	±SD	0.40	0.16	9.25	1.61	0.33	2.63
	n	5	5	5	5	5	5
G4, F & 1000	Mean	4.06	0.92	24.11	142.62	3.86	107.06
	±SD	0.50	0.09	7.55	1.62	0.40	3.20
	n	5	5	5	5	5	5

F: Female; SD: Standard Deviation; n: Number of Animals (randomly selected).

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin
		(GLU)	-	(CRE)	(CHO)	(TRI)	(TPR)	(ALB)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)
G1R, M & 0	Mean	99.80	27.36	0.55	59.60	23.00	6.98	3.10
	±SD	6.53	3.92	0.02	16.21	5.52	0.26	0.07
	n	5	5	5	5	5	5	5
G4R, M & 1000	Mean	99.40	25.74	0.56	68.80	31.60	7.12	3.05
	±SD	10.53	1.19	0.03	8.76	10.78	0.11	0.04
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	120.40	35.68	0.61	106.40	49.60	8.44	4.10
	±SD	7.16	7.57	0.02	17.07	34.09	0.56	0.18
	n	5	5	5	5	5	5	5
G4R, F & 1000	Mean	104.00*	31.42	0.59	89.20	41.60	8.04	3.70
	±SD	13.49	2.25	0.01	16.98	11.87	0.42	0.44
	n	5	5	5	5	5	5	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Alanine aminotransferase	Aspartate aminotransferase	Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous
		(ALT)	(AST)	(ALP)	(BIT)	(CAL)	(PHO)
		(U/L)	(U/L)	(U/L)	(mg/dL)	(mg/dL)	(mg/dL)
G1R, M & 0	Mean	52.00	101.80	99.00	0.02	10.42	5.72
	±SD	40.34	53.17	23.40	0.02	0.37	0.34
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	42.60	98.40	105.20	0.02	10.02	5.26
	±SD	9.86	18.49	29.84	0.01	0.15	0.50
	n	5	5	5	5	5	5
G1R, F & 0	Mean	54.00	94.00	70.80	0.02	11.20	4.20
	±SD	28.82	36.89	40.92	0.02	0.45	0.38
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	53.80	103.20	69.00	0.04	10.90	4.42
	±SD	21.53	39.30	60.10	0.02	0.37	0.18
	n	5	5	5	5	5	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 17 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/kg body weight/day)		Globulin	Albumin/ Globulin ratio	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(GLO)	(A/G Ratio)	(BUN)	(Na)	(K)	(CLO)
		(g/dL)	-	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1R, M & 0	Mean	3.88	0.80	12.77	142.82	3.56	105.48
	±SD	0.27	0.06	1.83	0.36	0.41	0.92
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	4.07	0.75	12.01	143.16	3.59	108.06*
	±SD	0.15	0.04	0.56	0.68	0.41	1.65
	n	5	5	5	5	5	5
G1R, F & 0	Mean	4.34	0.95	16.65	142.62	3.45	105.76
	±SD	0.42	0.08	3.53	0.94	0.17	1.52
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	4.34	0.85	14.66	142.26	3.62	106.40
	±SD	0.21	0.12	1.05	1.41	0.48	2.88
	n	5	5	5	5	5	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 18.     SUMMARY OF URINALYSIS RECORD

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1, M & 0	G2, M & 111	G3, M & 333	G4, M & 1000
	Number of Animals (randomly selected)		5	5	5	5
Physical Examination	Color	Pale Yellow	5	5	5	5
	Appearance	Clear	5	5	5	5
	Volume (mL)	Mean	10.6	10.2	11.0	11.4
		±SD	0.7	1.4	0.8	0.4
Chemical Examination	pH	Mean	7.5	7.4	7.4	7.5
		±SD	0.0	0.4	0.5	0.0
	Specific Gravity	Mean	1.008	1.011	1.009	1.009
		±SD	0.003	0.002	0.002	0.002
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.2
		±SD	0.0	0.0	0.0	0.0
	Bilirubin (mg/dL)	Neg	5	5	5	5
	Ketones (mg/dL)	Neg	2	-	2	1
		5	3	5	3	4
	Blood (Ery/µL)	Neg	5	4	5	5
		Ca80	-	1	-	-
	Protein (mg/dL)	Neg	3	-	3	1
		Trace	2	4	1	4
		30	-	1	1	-
	Nitrite	Neg	4	4	5	1
		Pos	1	1	-	4
	Leucocytes	Neg	3	-	3	1
		Ca15	1	1	1	2
		Ca70	1	3	1	2
		Ca125	-	1	-	-
	Glucose (mg/dL)	Neg	5	5	5	5
	Microalbumin (mg/dL)	Neg	-	-	2	1
		>15	2	5	2	4
		15	3	-	1	-
Microscopic Examination	Epi Cells	0	3	4	2	4
		0-1	1	1	2	1
		1-2	1	-	1	-
	Casts	Absent	4	4	4	5
		Present	1	1	1	-
	Crystals	Present	5	5	5	5

M: Male; SD: Standard Deviation; Pos: Positive; Neg: Negative; Ca: Calculated.
 >=: Greater than or equal to; >: Greater than.

TABLE 18 (Contd…). SUMMARY OF URINALYSIS RECORD

Examination	Group, Sex & Dose (mg/kg body weight/day)		G1R, M & 0	G4R, M &1000	G1R, F & 0	G4R, F & 1000
	Number of Animals (randomly selected)		5	5	5	5
Physical Examination	Color	Pale Yellow	2	5	3	3
		Yellow	3	-	2	2
	Appearance	Clear	3	2	4	4
		Turbid	2	3	1	1
	Volume (mL)	Mean	6.4	8.6	7.9	7.8
		±SD	1.7	3.1	2.2	0.8
Chemical Examination	pH	Mean	6.7	7.3	6.8	7.6
		±SD	0.9	0.4	1.0	1.0
	Specific Gravity	Mean	1.020	1.014	1.014	1.012
		±SD	0.007	0.002	0.007	0.008
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.6
		±SD	0.0	0.0	0.0	0.8
	Bilirubin (mg/dL)	Neg	2	5	5	2
		1	3	-	-	3
	Ketones (mg/dL)	Neg	1	2	5	5
		5	4	3	-	-
	Blood (Ery/µL)	Neg	5	5	5	4
		Ca10	-	-	-	1
	Protein (mg/dL)	Neg	2	5	4	2
		Trace	2	-	1	-
		30	1	-	-	-
		100	-	-	-	1
		>=300	-	-	-	2
	Nitrite	Neg	3	5	5	5
		Pos	2	-	-	-
	Leucocytes	Neg	-	1	3	2
		Ca15	1	4	2	-
		Ca70	4	-	-	3
	Glucose (mg/dL)	Neg	5	5	5	5
	Microalbumin (mg/dL)	Neg	-	1	3	1
		>15	3	-	1	3
		15	2	4	1	1
Microscopic Examination\	Epi Cells	0	3	3	2	2
		0-1	1	2	2	1
		1-2	1	-	1	-
		2-3	-	-	-	2
	Casts	Absent	5	2	5	4
		Present	-	3	-	1
	Crystals	Absent	-	2	1	2
		Present	5	3	4	3

M: Male; F: Female; R: Recovery; SD: Standard Deviation; Pos: Positive; Neg: Negative.                              
Ca: Calculated; >=: Greater than or equal to; >: Greater than.

TABLE 19.     SUMMARY OF VAGINAL SMEAR EXAMINATION RECORD FOR DETERMINATION OF OESTRUS CYCLICITY

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 Oestrus Cycle (Days)
G1, F & 0	12	n	11	1	Mean	4.65
		%	91.7	8.3	±SD	0.41
					n	12
G2, F & 111	12	n	11	0	Mean	4.43
		%	91.7	0.0	±SD	0.42
					n	12
G3, F & 333	12	n	12	0	Mean	4.68
		%	100.0	0.0	±SD	0.27
					n	12
G4, F & 1000	12	n	11	1	Mean	4.53
		%	91.7	8.3	±SD	0.34
					n	12

F: Female; SD: Standard Deviation; n: Number of Animals.

TABLE 20.     SUMMARY OF DELIVERY AND LITTER OBSERVATIONS RECORD

Group, Sex & Dose (mg/kg body weight/day)		Delivery Record At birth
		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	12.18		6.27	5.73	12.00		0.18	0.00	0.18		0.00	0.00	0.00	0.00		1.14		98.86
	±SD	2.68		1.90	1.35	2.41		0.40	0.00	0.40		0.00	0.00	0.00	0.00		0.42		2.54
	n	11		11	11	11		11	11	11		11	11	11	11		11		11
G2, F & 111	Mean	12.20		6.00	6.00	12.00		0.10	0.10	0.20		0.00	0.00	0.00	0.00		1.12		98.70
	±SD	3.29		1.94	1.89	3.09		0.32	0.32	0.42		0.00	0.00	0.00	0.00		0.57		2.76
	n	10		10	10	10		10	10	10		10	10	10	10		10		10
G3, F & 333	Mean	10.90		5.30	5.50	10.80		0.10	0.00	0.10		0.00	0.00	0.00	0.00		1.00		99.41
	±SD	2.77		2.00	1.27	2.53		0.32	0.00	0.32		0.00	0.00	0.00	0.00		0.39		1.86
	n	10		10	10	10		10	10	10		10	10	10	10		10		10
G4, F & 1000	Mean	12.82		6.36	6.09	12.45		0.18	0.18	0.36		0.00	0.00	0.00	0.00		1.16		96.57
	±SD	1.99		2.11	1.64	2.54		0.40	0.60	0.92		0.00	0.00	0.00	0.00		0.71		9.11
	n	11		11	11	11		11	11	11		11	11	11	11		11		11

F: Female; SD: Standard Deviation; n: Number of Dams; m/f: Male/Female; %: Percentage.

TABLE 20 (Contd…). SUMMARY RECORD OF DELIVERY AND LITTER OBSERVATIONS

Group, Sex & Dose (mg/kg body weight/day)		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 Pups (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	6.27	5.73	12.00		0.00	0.00	0.00		0.00	0.00	0.00		1.14		100.00
	±SD	1.90	1.35	2.41		0.00	0.00	0.00		0.00	0.00	0.00		0.42		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G2, F & 111	Mean	6.00	6.00	12.00		0.00	0.00	0.00		0.00	0.00	0.00		1.12		100.00
	±SD	1.94	1.89	3.09		0.00	0.00	0.00		0.00	0.00	0.00		0.57		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
G3, F & 333	Mean	5.30	5.50	10.80		0.00	0.00	0.00		0.00	0.00	0.00		1.00		100.00
	±SD	2.00	1.27	2.53		0.00	0.00	0.00		0.00	0.00	0.00		0.39		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
G4, F & 1000	Mean	6.36	6.09	12.45		0.00	0.00	0.00		0.00	0.00	0.00		1.16		100.00
	±SD	2.11	1.64	2.54		0.00	0.00	0.00		0.00	0.00	0.00		0.71		0.00
	n	11	11	11		11	11	11		11	11	11		11		11

F: Female; SD: Standard Deviation; n: Number of Dams; m/f: Male/Female; %: Percentage.

TABLE 20 (Contd…). SUMMARY RECORD OF DELIVERY AND LITTER OBSERVATIONS

Group, Sex & Dose (mg/kg body weight/day)		Pups Sacrificed for Litter Standardization / Hormonal analysis (No.)				Live Pups (No.) on LD 4 after Litter Standardization / Hormonal analysis (No.)				LD 5 to 7												Sex Ratio (m/f) on LD 7		Pup Survival Index (%) [LD 5 to 7]
										Live Pups (No.)				Dead Pups (No.)				Cannibalized (No.)
		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total		Male	Female	Total
G1, F & 0	Mean	0.00	1.09	1.09		6.27	4.64	10.91		6.27	4.64	10.91		0.00	0.00	0.00		0.00	0.00	0.00		1.55		100.00
	±SD	0.00	1.04	1.04		1.90	1.36	1.58		1.90	1.36	1.58		0.00	0.00	0.00		0.00	0.00	0.00		0.86		0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11		11
G2, F & 111	Mean	0.00	1.20	1.20		6.00	4.80	10.80		6.00	4.80	10.80		0.00	0.00	0.00		0.00	0.00	0.00		1.37		100.00
	±SD	0.00	1.03	1.03		1.94	1.23	2.20		1.94	1.23	2.20		0.00	0.00	0.00		0.00	0.00	0.00		0.62		0.00
	n	10	10	10		10	10	10		10	10	10		10	10	10		10	10	10		10		10
G3, F & 333	Mean	0.00	0.70	0.70		5.30	4.80	10.10		5.30	4.80	10.10		0.00	0.00	0.00		0.00	0.00	0.00		1.21		100.00
	±SD	0.00	0.95	0.95		2.00	1.03	1.73		2.00	1.03	1.73		0.00	0.00	0.00		0.00	0.00	0.00		0.67		0.00
	n	10	10	10		10	10	10		10	10	10		10	10	10		10	10	10		10		10
G4, F & 1000	Mean	0.00	1.64	1.64		6.36	4.45	10.82		6.36	4.45	10.82		0.00	0.00	0.00		0.00	0.00	0.00		2.07		100.00
	±SD	0.00	0.81	0.81		2.11	1.57	1.99		2.11	1.57	1.99		0.00	0.00	0.00		0.00	0.00	0.00		2.39		0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11		11

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; m/f: Male/Female.

TABLE 20 (Contd…). SUMMARY RECORD OF DELIVERY AND LITTER OBSERVATIONS

Group, Sex & Dose (mg/kg body weight/day)		LD 8 to 13												Sex Ratio (m/f) on LD 13		Pup Survival Index (%) [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	6.27	4.64	10.91		0.00	0.00	0.00		0.00	0.00	0.00		1.55		100.00
	±SD	1.90	1.36	1.58		0.00	0.00	0.00		0.00	0.00	0.00		0.86		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G2, F & 111	Mean	6.00	4.80	10.80		0.00	0.00	0.00		0.00	0.00	0.00		1.37		100.00
	±SD	1.94	1.23	2.20		0.00	0.00	0.00		0.00	0.00	0.00		0.62		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
G3, F & 333	Mean	5.30	4.80	10.10		0.00	0.00	0.00		0.00	0.00	0.00		1.21		100.00
	±SD	2.00	1.03	1.73		0.00	0.00	0.00		0.00	0.00	0.00		0.67		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
G4, F & 1000	Mean	6.36	4.45	10.82		0.00	0.00	0.00		0.00	0.00	0.00		2.07		100.00
	±SD	2.11	1.57	1.99		0.00	0.00	0.00		0.00	0.00	0.00		2.39		0.00
	n	11	11	11		11	11	11		11	11	11		11		11

F: Female; SD: Standard Deviation; n: Number of Dams; LD: Lactation Day; m/f: Male/Female; %: Percentage.

TABLE 21.     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 Fertility Index (%)
G1, M & 0	12 (12)	100.0		11 (12)	91.7
G2, M & 111	12 (12)	100.0		10 (12)	83.3
G3, M & 333	12 (12)	100.0		10 (12)	83.3
G4, M & 1000	12 (12)	100.0		11 (12)	91.7

M: Male.

TABLE 21 (Contd…). SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE

Group, Sex & Dose (mg/kg body weight/day)		Cohabitation Record and Pre-coital Interval (Mean Time of Mating)					Gestational Length (duration of pregnancy)
		Pre-coital Interval (Days)		Conceiving Days (1 to 5)	Conceiving Days (More than 5 days)		Gestation Length (Days)
G1, F & 0	Mean	3.58	n	8	4		22.55
	SD	3.80	%	66.67	33.33		0.52
	N	12					11
G2, F & 111	Mean	3.75	n	9	3		22.20
	SD	4.45	%	75	25		0.63
	N	12					10
G3, F & 333	Mean	4.58	n	8	4		22.50
	SD	5.00	%	66.67	33.33		0.53
	N	12					10
G4, F & 1000	Mean	4.67	n	8	4		22.36
	SD	5.50	%	66.67	33.33		0.50
	N	12					11

F: Female; SD: Standard Deviation; n: Number of females with evidence of mating (pre-coital interval); n: Number of pregnant females (gestation length).

TABLE 21 (Contd…). SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)			Female Fertility 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 Fertility Index (%)
G1, F & 0	12 (12)	100.0		11 (12)	91.7
G2, F & 111	12 (12)	100.0		10 (12)	83.3
G3, F & 333	12 (12)	100.0		10 (12)	83.3
G4, F & 1000	12 (12)	100.0		11 (12)	91.7

F: Female.

TABLE 21 (Contd…). SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE

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 Parturition	No. of Females with Evidence of Pregnancy	Gestation Index (%)
G1, F & 0	11	12	91.7		11	11	100.0
G2, F & 111	10	12	83.3		10	10	100.0
G3, F & 333	10	12	83.3		10	10	100.0
G4, F & 1000	11	12	91.7		11	11	100.0

F: Female.

TABLE 21 (Contd…). SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE

Group, Sex & Dose  (mg/kg body weight/day)	Parturition index (%)
	No. of Females Littered	No. of Females with evidence of pregnancy	Parturition index (%)
G1, F & 0	11	11	100.0
G2, F & 111	10	10	100.0
G3, F & 333	10	10	100.0
G4, F & 1000	11	11	100.0

F: Female.

TABLE 21 (Contd…). SUMMARY RECORD OF REPRODUCTIVE PERFORMANCE

Group, Sex & Dose (mg/kg body weight/day)		Post-implantation Loss (%)					Post-natal Loss (%)
		No. of Implantations	No. of Viable (Live) Pups	Post-implantation Loss (No.)	Post-implantation Loss (%)		Total No. of Pups Found Dead/ Cannibalized during lactation period	Post-natal Loss (%)
G1, F & 0	Mean	12.45	12.00	0.45	3.70		0.00	0.00
	±SD	2.42	2.41	0.52	4.40		0.00	0.00
	n	11	11	11	11		11	11
G2, F & 111	Mean	12.70	12.00	0.70	6.02		0.00	0.00
	±SD	2.75	3.09	1.06	9.67		0.00	0.00
	n	10	10	10	10		10	10
G3, F & 333	Mean	11.00	10.80	0.20	1.50		0.00	0.00
	±SD	2.75	2.53	0.42	3.25		0.00	0.00
	n	10	10	10	10		10	10
G4, F & 1000	Mean	13.27	12.45	0.82	6.72		0.00	0.00
	±SD	1.74	2.54	1.40	11.56		0.00	0.00
	n	11	11	11	11		11	11

F: Female; n: Number of Dams.

TABLE 22.     SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain
G1, M & 0	Mean	0.0667	0.2891	0.7915	3.3315	1.4198	1.3943	2.9537	2.1193
	±SD	0.0045	0.0466	0.2701	0.3035	0.1420	0.2048	0.2510	0.0321
	n	5	5	5	12	12	5	5	5
G2, M & 111	Mean	0.0719	0.3589	0.8023	3.4284	1.4112	1.5033	2.8196	2.1336
	±SD	0.0094	0.0292	0.0928	0.1782	0.1365	0.1061	0.1723	0.0957
	n	5	5	5	12	12	5	5	5
G3, M & 333	Mean	0.0709	0.4450*	0.8041	3.3728	1.4195	1.4754	2.8317	2.1467
	±SD	0.0060	0.0665	0.1455	0.2771	0.1776	0.1560	0.1507	0.0446
	n	5	5	5	12	12	5	5	5
G4, M & 1000	Mean	0.0716	0.4129*	0.8155	3.3454	1.5221	1.5218	3.0082	2.2033
	±SD	0.0115	0.1013	0.1404	0.2101	0.1812	0.3979	0.7565	0.1355
	n	5	5	5	12	12	5	5	5

M: Male; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 22 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Liver	Prostate	Seminal vesicles with coagulating glands	Lungs	Pituitary	Thyroid along with parathyroid^#
G1, M & 0	Mean	10.0491	1.1499	1.6300	1.9467	0.0208	0.0296
	±SD	1.6192	0.1975	0.4468	0.1258	0.0014	0.0017
	n	5	12	12	5	5	12
G2, M & 111	Mean	10.3930	1.1988	1.6027	2.0659	0.0197	0.0290
	±SD	0.4389	0.2552	0.3454	0.1175	0.0013	0.0020
	n	5	12	12	5	5	12
G3, M & 333	Mean	10.6576	1.3430	1.7846	2.0478	0.0203	0.0301
	±SD	0.7376	0.3501	0.3700	0.1024	0.0009	0.0013
	n	5	12	12	5	5	12
G4, M & 1000	Mean	10.8065	1.2209	1.9124	2.0837	0.0205	0.0305
	±SD	2.8155	0.3931	0.5906	0.1492	0.0015	0.0023
	n	5	12	12	5	5	12

M: Male; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation.

TABLE 22 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
G1, F & 0	Mean	0.0741	0.2781	0.7048	0.1738	0.5901	1.1848
	±SD	0.0083	0.0317	0.0646	0.0299	0.1167	0.1262
	n	5	5	5	12	11^@	5
G2, F & 111	Mean	0.0884	0.2632	0.7503	0.1432*	0.5097	1.1940
	±SD	0.0110	0.0345	0.1289	0.0332	0.1138	0.0729
	n	5	5	5	12	11^@	5
G3, F & 333	Mean	0.0728	0.2267*	0.6584	0.1291*	0.5413	1.2001
	±SD	0.0094	0.0282	0.1051	0.0193	0.0886	0.0513
	n	5	5	5	12	12	5
G4, F & 1000	Mean	0.0775	0.2179*	0.6109	0.1466*	0.5283	1.3318
	±SD	0.0093	0.0063	0.0413	0.0190	0.1008	0.3482
	n	5	5	5	12	12	5

F: Female; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

@: One animal data from group G1 and G2 is excluded for mean calculations as the weight varies from normal uterus due to presence of dead foetus during necropsy, but the data is presented in individual animal data. (Appendix 22).

TABLE 22 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Lungs	Pituitary	Thyroid along with parathyroid^#
G1, F & 0	Mean	2.2668	2.0179	11.5641	1.2626	0.0179	0.0266
	±SD	0.1164	0.0746	0.5339	0.1185	0.0011	0.0015
	n	5	5	5	5	5	12
G2, F & 111	Mean	2.0463*	2.0056	12.5985	1.5260	0.0183	0.0272
	±SD	0.1081	0.1131	2.0359	0.2954	0.0014	0.0015
	n	5	5	5	5	5	12
G3, F & 333	Mean	2.0269*	1.9917	10.1148	1.3551	0.0176	0.0265
	±SD	0.0937	0.0552	1.8164	0.1082	0.0008	0.0015
	n	5	5	5	5	5	12
G4, F & 1000	Mean	1.9230*	2.0216	10.2708	1.3780	0.0180	0.0275
	±SD	0.0912	0.0446	1.3180	0.1188	0.0010	0.0014
	n	5	5	5	5	5	12

F: Female; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 22 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain
G1R, M & 0	Mean	0.0671	0.2367	0.8804	3.3055	1.6954	1.6355	2.2619	2.0448
	±SD	0.0080	0.0348	0.0907	0.1930	0.0763	0.1477	0.3680	0.1172
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.0638	0.2709	0.7442*	3.4656	1.6882	1.6050	2.3506	2.0483
	±SD	0.0051	0.0738	0.0528	0.1133	0.0690	0.3581	0.3464	0.1621
	n	5	5	5	5	5	5	5	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 22 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Liver	Prostate	Seminal vesicles with coagulating glands	Lungs	Pituitary	Thyroid along with parathyroid^#
G1R, M & 0	Mean	12.6730	1.3824	1.8300	2.0437	0.0207	0.0299
	±SD	1.2027	0.2475	0.4858	0.5420	0.0009	0.0015
	n	5	5	5	5	5	5
G4R, M & 1000	Mean	12.5717	1.6038	1.8342	1.9398	0.0210	0.0306
	±SD	1.2099	0.1130	0.4887	0.0584	0.0011	0.0008
	n	5	5	5	5	5	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation.

TABLE 22 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
G1R, F & 0	Mean	0.0706	0.2511	0.6464	0.1265	0.6499	1.0512
	±SD	0.0064	0.0443	0.0886	0.0164	0.0920	0.0882
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	0.0702	0.2467	0.6261	0.1543*	0.6334	1.0135
	±SD	0.0121	0.0337	0.1113	0.0105	0.0853	0.0746
	n	5	5	5	5	5	5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 22 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Lungs	Pituitary	Thyroid along with parathyroid^#
G1R, F & 0	Mean	1.9138	2.0075	9.6308	1.8480	0.0185	0.0278
	±SD	0.0978	0.0986	0.9760	0.0946	0.0009	0.0014
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	1.8218	1.9767	8.6614	2.0609	0.0193	0.0271
	±SD	0.1551	0.0844	1.7719	0.4014	0.0014	0.0006
	n	5	5	5	5	5	5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation.

TABLE 23.     SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart
G1, M & 0	Mean	440.14	0.0150	0.0648	0.1748	0.7637	0.3257	0.3099
	±SD	38.09	0.0018	0.0115	0.0484	0.1095	0.0493	0.0251
	n	12	5	5	5	12	12	5
G2, M & 111	Mean	433.53	0.0162	0.0812	0.1809	0.7928	0.3262	0.3390
	±SD	29.89	0.0021	0.0090	0.0188	0.0447	0.0319	0.0185
	n	12	5	5	5	12	12	5
G3, M & 333	Mean	437.73	0.0159	0.0995*	0.1797	0.7734	0.3250	0.3314
	±SD	26.65	0.0016	0.0135	0.0302	0.0792	0.0413	0.0427
	n	12	5	5	5	12	12	5
G4, M & 1000	Mean	443.94	0.0156	0.0891*	0.1779	0.7570	0.3432	0.3304
	±SD	38.78	0.0013	0.0124	0.0187	0.0592	0.0298	0.0626
	n	12	5	5	5	12	12	5

M: Male; SD: Standard Deviation; n: Number of Animals.

TABLE 23 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Prostate	Seminal vesicles with coagulating glands	Lungs	Pituitary	Thyroid along with parathyroid^#
G1, M & 0	Mean	0.6591	0.4745	2.2336	0.2614	0.3768	0.4360	0.0047	0.0067
	±SD	0.0371	0.0334	0.2239	0.0389	0.1233	0.0428	0.0003	0.0004
	n	5	5	5	12	12	5	5	12
G2, M & 111	Mean	0.6380	0.4818	2.3449	0.2758	0.3681	0.4668	0.0044	0.0067
	±SD	0.0606	0.0272	0.0585	0.0505	0.0681	0.0350	0.0003	0.0006
	n	5	5	5	12	12	5	5	12
G3, M & 333	Mean	0.6350	0.4812	2.3924	0.3052	0.4081	0.4594	0.0046	0.0069
	±SD	0.0467	0.0237	0.2415	0.0710	0.0824	0.0376	0.0001	0.0004
	n	5	5	5	12	12	5	5	12
G4, M & 1000	Mean	0.6545	0.4865	2.3426	0.2767	0.4271	0.4575	0.0045	0.0069
	±SD	0.1286	0.0618	0.4199	0.0940	0.1161	0.0285	0.0002	0.0004
	n	5	5	5	12	12	5	5	12

M: Male; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation.

TABLE 23 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
G1, F & 0	Mean	305.54	0.0242	0.0912	0.2307	0.0571	0.1938	0.3872
	±SD	21.99	0.0035	0.0163	0.0360	0.0100	0.0422	0.0618
	n	12	5	5	5	12	11^@	5
G2, F & 111	Mean	314.85	0.0286	0.0853	0.2428	0.0458*	0.1635	0.3854
	±SD	19.21	0.0044	0.0142	0.0457	0.0115	0.0448	0.0220
	n	12	5	5	5	12	11^@	5
G3, F & 333	Mean	302.78	0.0236	0.0730	0.2142	0.0431*	0.1801	0.3879
	±SD	22.49	0.0038	0.0045	0.0429	0.0092	0.0352	0.0151
	n	12	5	5	5	12	12	5
G4, F & 1000	Mean	306.64	0.0245	0.0690*	0.1938	0.0483	0.1749	0.4241
	±SD	25.44	0.0033	0.0062	0.0238	0.0080	0.0430	0.1222
	n	12	5	5	5	12	12	5

F: Female; SD: Standard Deviation; n: Number of Animals.

@: One animal data from group G1 and G2 is excluded for mean calculations as the weight varies from normal uterus due to presence of dead foetus during necropsy, but the data is presented in individual animal data. (Appendix 23).

TABLE 23 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Lungs	Pituitary	Thyroid along with parathyroid^#
G1, F & 0	Mean	0.7409	0.6583	3.7703	0.4138	0.0058	0.0087
	±SD	0.0909	0.0663	0.3600	0.0696	0.0003	0.0005
	n	5	5	5	5	5	12
G2, F & 111	Mean	0.6618	0.6488	4.0668	0.4903	0.0059	0.0087
	±SD	0.0563	0.0584	0.6665	0.0770	0.0002	0.0009
	n	5	5	5	5	5	12
G3, F & 333	Mean	0.6560	0.6443	3.2646	0.4388	0.0057	0.0088
	±SD	0.0476	0.0330	0.5652	0.0452	0.0002	0.0007
	n	5	5	5	5	5	12
G4, F & 1000	Mean	0.6097*	0.6402	3.2355	0.4353	0.0057	0.0090
	±SD	0.0642	0.0516	0.3329	0.0373	0.0008	0.0007
	n	5	5	5	5	5	12

F: Female; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 23 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart
G1R, M & 0	Mean	496.58	0.0136	0.0476	0.1785	0.6684	0.3430	0.3320
	±SD	40.88	0.0022	0.0051	0.0257	0.0554	0.0286	0.0478
	n	5	5	5	5	5	5	5
G4R, M & 1000	Mean	502.58	0.0127	0.0538	0.1482*	0.6902	0.3358	0.3184
	±SD	12.09	0.0010	0.0137	0.0126	0.0356	0.0088	0.0643
	n	5	5	5	5	5	5	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 23 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Prostate	Seminal vesicles with coagulating glands	Lungs	Pituitary	Thyroid along with parathyroid^#
G1R, M & 0	Mean	0.4554	0.4147	2.5665	0.2783	0.3746	0.4088	0.0042	0.0060
	±SD	0.0586	0.0488	0.3239	0.0415	0.1185	0.0840	0.0002	0.0003
	n	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	0.4682	0.4080	2.4984	0.3193	0.3643	0.3862	0.0042	0.0061
	±SD	0.0730	0.0361	0.1858	0.0248	0.0941	0.0153	0.0002	0.0003
	n	5	5	5	5	5	5	5	5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of Animals; #: Weighed post fixation.

TABLE 23 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart
G1R, F & 0	Mean	308.17	0.0229	0.0822	0.2106	0.0410	0.2112	0.3432
	±SD	16.88	0.0016	0.0185	0.0336	0.0043	0.0292	0.0498
	n	5	5	5	5	5	5	5
G4R, F & 1000	Mean	311.82	0.0225	0.0791	0.2015	0.0495*	0.2026	0.3256
	±SD	12.71	0.0037	0.0102	0.0405	0.0029	0.0205	0.0293
	n	5	5	5	5	5	5	5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 23 (Contd…). SUMMARY OF TERMINAL BODY WEIGHT (g) AND ORGAN WEIGHT RELATIVE TO
TERMINAL BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Kidneys	Brain	Liver	Lungs	Pituitary	Thyroid along with parathyroid^#
G1R, F & 0	Mean	0.6217	0.6518	3.1221	0.6009	0.0060	0.0090
	±SD	0.0295	0.0166	0.2213	0.0411	0.0002	0.0005
	n	5	5	5	5	5	5
G4R, F & 1000	Mean	0.5840	0.6342	2.7664	0.6616	0.0062	0.0087
	±SD	0.0386	0.0217	0.4774	0.1320	0.0003	0.0002
	n	5	5	5	5	5	5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of Animals.

TABLE 24.     SUMMARY OF SERUM T4 HORMONE LEVELS (ng/mL) RECORD - ADULTS

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	78.813
	±SD	7.616
	n	12
G2, M & 111	Mean	67.002*
	±SD	12.097
	n	12
G3, M & 333	Mean	79.275
	±SD	10.083
	n	12
G4, M & 1000	Mean	77.258
	±SD	13.137
	n	12

M: Male; SD: Standard Deviation; n: Number of Animals; *: Statistically significant (P<0.05) change than the vehicle control group.

TABLE 25.     SUMMARY RECORD OF PUP OBSERVATIONS DURING POSTNATAL 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	6	11	11
	No. of Live Pups	132	132	11	120	120
	Pup Observation/ No. of Pups observed	N/132	N/132	-	N/120	N/120
G2, F & 111	No. of Dams^#	10	10	6	10	10
	No. of Live Pups	120	120	12	108	108
	Pup Observation/ No. of Pups observed	N/120	N/120	-	N/108	N/108
G3, F & 333	No. of Dams^#	10	10	4	10	10
	No. of Live Pups	108	108	7	101	101
	Pup Observation/ No. of Pups observed	N/108	N/108	-	N/101	N/101
G4, F & 1000	No. of Dams^#	11	11	9	11	11
	No. of Live Pups	137	137	18	119	119
	Pup Observation/ No. of Pups observed	N/137	N/137	-	N/119	N/119

F: Female; N: Normal; PND: Postnatal Day; #: Dams confirmed with littering.

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

TABLE 26.     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	6.79	6.23		10.88	10.35		14.87	14.37		28.44	27.57
	±SD	0.04	0.08		0.12	0.10		0.11	0.13		0.36	0.46
	n	11 (69)	11 (63)		11 (69)	11 (63)		11 (69)	11 (51)		11 (69)	11 (51)
G2, F & 111	Mean	6.79	6.28		10.89	10.35		14.88	14.39		28.29	27.56
	±SD	0.08	0.14		0.12	0.11		0.17	0.21		0.37	0.51
	n	10 (60)	10 (60)		10 (60)	10 (60)		10 (60)	10 (48)		10 (60)	10 (48)
G3, F & 333	Mean	6.84	6.30		10.91	10.37		14.91	14.42		28.50	27.90
	±SD	0.08	0.09		0.10	0.12		0.15	0.17		0.46	0.42
	n	10 (53)	10 (55)		10 (53)	10 (55)		10 (53)	10 (48)		10 (53)	10 (48)
G4, F & 1000	Mean	6.79	6.23		10.87	10.32		14.88	14.36		28.31	27.39
	±SD	0.06	0.07		0.09	0.11		0.12	0.17		0.44	0.37
	n	11 (70)	11 (67)		11 (70)	11 (67)		11 (70)	11 (49)		11 (70)	11 (49)

F: Female; SD: Standard Deviation; n: Number of Litters (number of pups); PND: Postnatal Day.

TABLE 27.     SUMMARY RECORD OF MEAN PUP ANOGENITAL DISTANCE (AGD) MEASUREMENT (mm) AND ANOGENITAL DISTANCE (AGD) RATIO PER LITTER ON POSTNATAL DAY 4

Group & Dose (mg/kg body weight/day)		AGD Measurement (mm)			AGD Ratio
		Male	Female		Male	Female
G1, F & 0	Mean	4.81	2.45		2.17	1.13
	±SD	0.12	0.08		0.05	0.03
	n	11 (69)	11 (63)		11 (69)	11 (63)
G2, F & 111	Mean	4.89	2.45		2.21	1.12
	±SD	0.09	0.08		0.03	0.03
	n	10 (60)	10 (60)		10 (60)	10 (60)
G3, F & 333	Mean	4.95*	2.46		2.23*	1.13
	±SD	0.07	0.03		0.03	0.02
	n	10 (53)	10 (55)		10 (53)	10 (55)
G4, F & 1000	Mean	4.89	2.50		2.21*	1.15
	±SD	0.09	0.09		0.04	0.04
	n	11 (70)	11 (67)		11 (70)	11 (67)

F: Female; SD: Standard Deviation; n: Number of Litters (number of pups); AGD: Anogenital Distance.

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

TABLE 28.     SUMMARY RECORD OF OBSERVATION OF MALE PUPS FOR RETENTION OF NIPPLE/AREOLAE (No.) RECORD

Group & Dose (mg/kg body weight/day)		Mean No. of Pups with Retention of Nipples/ Areolae on Postnatal Day 13
G1 & 0	Mean	0.00
	±SD	0.00
	n	11 (69)
G2 & 111	Mean	0.00
	±SD	0.00
	n	10 (60)
G3 & 333	Mean	0.00
	±SD	0.00
	n	10 (53)
G4 & 1000	Mean	0.00
	±SD	0.00
	n	11 (70)

F: Female; SD: Standard Deviation; n: Number of Litters (number of male pups).

TABLE 29.     SUMMARY OF SERUM T4 HORMONE LEVELS (ng/mL) RECORD - POSTNATAL DAY (PND) 13 PUPS

Group & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, F & 0	Mean	71.557
	±SD	6.196
	n	11
G2, F & 111	Mean	58.207*
	±SD	6.124
	n	10
G3, F & 333	Mean	55.550*
	±SD	3.863
	n	10
G4, F & 1000	Mean	60.962*
	±SD	6.316
	n	11

F: Female; SD: Standard Deviation; n: Number of Litters (number of pups); PND: Postnatal Day

Note: No pups were sacrificed from group G4, as the litter size is equal to or less than 10.

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

Conclusions:

The oral administration of test item over the time of the study, carried out according to the OECD guideline 422 (for main group males with a total of 31 days, for main group females ranging from 50 to 62 days and for recovery group animals with a total period of 52 days) did not produce any indication of systemic, reproduction and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of test item is considered as 1000 mg/kg body weight for systemic, reproduction and developmental toxicity end points.

Executive summary:

The objective of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by oral gavage in Sprague Dawley Rats was to determine the possible health hazards likely to arise from repeated exposure to the test item over a relatively limited period of time. This study was also conducted to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 116 (58 males + 58 females) Sprague Dawley rats were selected for the study and distributed to four main (G1, G2, G3 and G4) and two recovery groups (G1R and G4R). Each main group consisted of 12 males and 12 females and each recovery group consisted of 5 males and 5 females. The animals in group G1/G1R were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], animals in groups G2, G3 and G4/G4R were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight/day for low, mid and high/high dose recovery, respectively. Vehicle and test item formulations were administered orally by gavage with the dose volume of 10 mL/kg body weight/day once daily at similar times on each day for 7 days a week.

All the main group males were administered with test item during pre-mating (14 -days), during mating and during post-mating period (total of 31 days). The pregnant females from main groups were administered with test item during pre-mating (14 -days), during mating, pregnancy (gestation) and up to lactation day 13. The females with positive mating signal, but no-evidence of pregnancy / littering were administered with test item during pre-mating (14 -days), during mating, and further 24 days from the day of positive mating signal. All the recovery group animals were administered with test item for 50 days followed by a 14 -day observation period without treatment.

The test item formulations were stable for 6 hours and 48 hours at room temperature in 0.5% w/v Carboxymethyl Cellulose with the concentrations of 5 mg/mL and 100 mg/mL. The homogeneity and dose form concentration analysis for prepared test item formulations during treatment period was performed during week 1 and 5 and the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is less than 15%.

For systemic toxicity assessment, all the animals (main and recovery groups) of both sexes were observed once daily for clinical signs, twice daily for mortality and morbidity and once in a week for detailed clinical examination. Body weight of all the animals (main and recovery groups) of both sexes were recorded once in a week until termination. Further, the body weight of all pregnant females was recorded on gestation day (GD) 0, 7, 14 and 20 during pregnancy and on lactation day (LD) 1, 4, 7, 13 and 14 during lactation. Body weight of all non-pregnant females was recorded once in a week until termination. Feed consumption for all the animals of both sexes were recorded once in a week during pre-mating for main groups and throughout the experimental period for recovery groups. Further, feed consumption of all pregnant females was recorded during GD 0 to 7, 7 to 14 and 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13. Ophthalmological examination was carried out once before treatment and at the end of the dosing period for all males and females (main and recovery groups).

Neurological/functional observations were performed for five randomly selected males and females from each main group, for all animals from recovery groups towards end of the dosing period. The investigations of haematology and clinical chemistry for all animals (main and recovery groups), investigations of urinalysis for five randomly selected males from each main group and for all recovery groups animals was conducted at termination. Serum thyroxine hormone (T4) levels were estimated for all main group males by ELISA. The organs were collected and weighed on the day of termination for all animals (main and recovery groups) of both sexes and organ weight relative to terminal body weight was calculated. All the animals (main and recovery groups) of both sexes were observed for both external and internal gross pathological changes during conduct of necropsy. Histopathological examination was conducted on all the tissues collected from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

For reproductive toxicity assessment, all the males (main groups) were evaluated for reproductive performances such as, mating and fertility indices. All the females (main groups) were evaluated for reproductive performances such as, mating, fertility, gestation and parturition indices. The females were also evaluated for pre-coital interval and gestation length. All females were evaluated for oestrus cyclicity during pre-mating period and the vaginal smear examination was continued until evidence of mating. At birth (delivery) parameters such as, number of live/dead pups born, litter size, sex ratio (m/f) and live birth index per litter were observed / calculated. The litter observations during lactation period such as, number of survived or dead pups, sex ratio (m/f) and pup survival index per litter were observed / calculated. The total number of implantation sites was recorded for each litter during necropsy and the post-implantation and postnatal losses per litter was calculated.

For developmental toxicity assessment, all survived pups from each litter were observed once daily for external behavioural changes and twice daily for mortality until termination [postnatal day (PND) 13]. All pups from each litter were weighed individually on PND 1, 4, 7 and 13 and measured for anogenital distance on PND 4. All male pups were observed for retention of nipples on PND 13. All the pups were subjected for both external and internal gross pathological changes during conduct of necropsy. The collected serum from PND 13 pups was subjected to estimation of thyroxine hormone (T4) levels using ELISA.

There were no clinical signs of toxicity and no mortality/morbidity noted in any of the animals from all the tested dose groups, however dark purple colored faeces were noted from day 2 observation throughout the experiment period till termination of treatment in group G3 & G4/G4R animals. This coloration is due to colored nature of the test item but not any adverse sign. Detailed clinical examination did not reveal any changes in any of the tested dose group animals. No changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption in all the tested dose groups of both sexes. The ophthalmological examination and neurological/functional examinations did not reveal any test item-related changes in any of the animals from all the tested dose groups of both sexes. The obtained mean haematology, clinical chemistry and urinalysis values did not reveal any test item-related changes in all the tested dose groups. The estimated serum T4 levels did not reveal any changes. The absolute and relative organ weights of both sexes did not reveal any changes from all the tested dose groups.

No treatment related gross pathological changes were noted in any of the animals from all the tested dose groups during necropsy, however in test item administered animals of both sexes, the contents of gastrointestinal tract were found to be violet in color which was attributed to the color of the test item and was considered as non-adverse effect as the mucosa of gastrointestinal tract was found to be within normal limits.

No treatment related microscopic findings noted in any of the tissues/organs subjected for histopathological evaluation from both high dose group and vehicle control group animals of both sexes.  

There were no effects noted on reproductive performance of both sexes in all the tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups during treatment period. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all the tested dose groups. No effects were noted for live birth index and pup survival index in all the tested dose groups. No test item-related post-implantation losses were noted in all the tested dose groups.

There were no developmental/external behavioural changes noted and no test item-related mortalities noted during postnatal period in any of pups from all the tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were un-affected by the test item in all the tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all the tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all the tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested dose and control group litters.

Reference 4

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Reason / purpose for cross-reference:

data waiving: supporting information

Remarks:

Doses / Concentrations:

Basis:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

reliable without restriction

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

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Based on the obtained results from this pre-natal developmental toxicity study conducted according to OECD TG 414 in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of the test item for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Remarks:

Prenatal Developmental Toxicity Study in Rats

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May 2021 to 27 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: Dose range finding study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxy Methyl Cellulose within the mean percent recovery range of 85 to 115
- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulation
- Final preparation of a solid: Suspension

FORM AS APPLIED IN THE TEST: Liquid suspension

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on
Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA
- Age at study initiation: Minimum 9 weeks
- Weight at study initiation: Males: 260.41 to 287.31 g and Females: 212.17 to 270.12 g
- Housing: Housed in sterilized standard polypropylene cage (Size: L 430 × B 285 × H 150 mm).
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: Start: 28 May 2021 to 23 June 2021.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.4
- Humidity (%): 47 to 64
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 28 May 2021 To: 19 July 2021

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5 % w/v

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and uniformly suspended in 0.5% w/v Carboxy Methyl Cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight) as per in-house solubility/suspendibility test results. Hence, 0.5% w/v Carboxy Methyl Cellulose was used as vehicle for test item formulations in consultation with sponsor.
0.5% w/v Carboxy Methyl Cellulose is a routinely used and universally accepted
vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: G2: 11.1 mg/mL; G3: 33.3 mg/mL; G4: 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
- batch no.: SLCD3996

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis for dose concentration verification was done by Analytical Chemistry department of Bioneeds India Private Limited. Sampling and analysis of formulations were performed during first week and last week of the treatment. Approximately, 5 mL of 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. The collected samples were transferred to Analytical Chemistry department of
Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at
established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. 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) is ≤15%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2 (M:F)
- Length of cohabitation: 14 days initially + 7 days extention
- 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: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19 [15 days per animal]

Frequency of treatment:

once daily

Duration of test:

15 days per animal starting from implnatation to one day prior to scheduled delivery

Dose / conc.:

111 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

25 presumed pregnant females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item Hostaperm Red Violet ER 02. However, the NOAEL of Pigment Red 282, the structural analogue of test item is 1000 mg/kg body weight/day estimated in a 28-Day Repeated Dose Oral Toxicity in Wistar Rats performed according to OECD TG 407, (https://echa.europa.eu/registration-dossier/-/registereddossier/14755/7/6/2#).

Based on the above available information, the doses of 0, 111, 333 and 1000 mg/kg body weight/day had been selected as control, low, mid and high dose groups for the dose range finding study (BIO-DTX 056) in consultation with the sponsor.
There were no test item-related related changes noted in any of the systemic, reproduction and developmental parameters up to the dose level of 1000 mg/kg body weight/day in the Dose Range Finding Study (BIO-DTX 056).

Hence, the same doses of 0, 111, 333 and 1000 mg/kg body weight/day were selected as control, low, mid and high dose groups respectively, for present prenatal developmental toxicity study in consultation with the sponsor

- Rationale for animal assignment (if not random): The body weight of mated females on each day of gestation day ‘0’ was recorded and arranged in the ascending order of their body weight until required number of mated females acquired for each group. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weight for all groups and permanent identification numbers were assigned

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations checked in table [No.2] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:Gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and
on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All visceral organs along with uterus and ovaries.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]

Statistics:

The raw data was subjected to computer statistical processing. The computer printout of the data (in the form of appendix) was verified with the raw data. After verification, the data was subjected to various statistical analysis using SPSS software version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests was designated by the superscripts throughout the report as, * Statistically significant (P<0.05) change than the control group.

Parametric: One-way ANOVA with Dunnett’s post test: Maternal body weight, Percent change in maternal body weight,Corrected body weight for maternal increase,Gravid uterus weight,Maternal Feed consumption, Mean fetal weight per dam, Mean fetal crown rump length per dam, Mean fetal anogenital distance per dam, Serum T3, T4 and TSH levels and Organ weight.

Non-Parametric:Kruskal-Wallis test : No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live fetuses per dam, Percent of live fetuses per dam, No. of early/late resorptions per dam,Percent of early/late resorptions per dam,Sex ratio (m/f) per dam,Pre/Post implantation losses (%) per dam,Fetal external / visceral / skeletal anomalies per dam.

Frequencies Comparison (Cross Tabs): Chi-square test: Frequencies comparison for No. of pregnant / non-pregnant,No. of dams with / without live fetuses,No. of dams with / without dead fetuses,No. of dams with / without resorptions.

Indices:

Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight

Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation sites / Total Number of Corpora lutea x 100

Post-implantation Loss (%) = Number of Implantation sites - Number of Viable Fetuses / Number of Implantation Sites X 100

Anogenital Distance (AGD) Ratio = Anogenital Distance (mm) / Cube root of fetal weight x 100

Historical control data:

Included in report where applicable

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related histopathological findings noted in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats. (Elizabeth McInnes, 2012).

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The mean serum T3 levels (ng/mL) were 1.567, 1.617, 1.827 and 2.092 for groups G1, G2, G3 and G4 respectively. There were no test item-related changes noted for this parameter across the dose groups when compared to the vehicle control group. The noted statistically significant increase in mean serum T3 levels (ng/mL) in groups G3 and G4 is considered as incidental and un-related to treatment with test item as the obtained mean values are within normal range of same species and strain.

The mean serum T4 levels (ng/mL) were 52.581, 51.869, 52.053 and 54.421 for groups G1, G2, G3 and G4 respectively. There were no changes noted for this parameter across the dose groups when compared to the vehicle control group.

The serum TSH levels (µIU/mL) were 1.906, 1.942, 1.870 and 1.370 for groups G1, G2, G3 and G4 respectively. There were no changes for this parameter across the dose groups when compared to the vehicle control group.

Details on results:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of general/systemic toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions noted

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-Implantation Loss: The mean percentage of pre-implantation loss per litter was 4.71, 0.00, 1.43 and 0.00 for groups G1, G2, G3 and G4 respectively.
There was no test item related changes noted for percentage of pre-implantation loss per litter in all the tested dose groups. The noted statistically significant reduction in percentage of pre-implantation loss per litter in groups G2 and G4 when compared to the vehicle control group is considered as toxicologically irrelevant.

Post-Implantation Loss: The mean percentage of post-implantation loss per litter was 5.67, 3.17, 2.64 and 5.18 for G1, G2, G3 and G4 respectively.
There were no changes or no statistically significant differences noted for mean percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G4.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 - ca. 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

maternal abnormalities

mortality

necropsy findings

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

other: Serum T3, T4 and TSH levels

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Localisation:

amniotic fluid

cervix

mammary gland

ovary

oviduct

placenta

umbilical cord

uterus

vagina

vitreous chamber / humor

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal external alterations were noted across the dose groups and vehicle control group (refer table no. 15). These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal skeletal alterations were noted across the dose groups and vehicle control group (refer table no. 17). These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal soft tissue/visceral alterations were noted across the dose groups and vehicle control group (refer table no. 16). These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

The oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 1 000 - ca. 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

visceral malformations

other: Anogenital distance and Crown rump length

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

external: cranium

external: ear

external: eye

external: face

external: limb

external: paw

external: tail

external: trunk

external: anogenital distance

external: anus

external: genital tubercle

external: large intestine

external: thorax

external: umbilicus

external: pelvic region

skeletal: skull

skeletal: skull, fontanelles

skeletal: skull sutures

skeletal: clavicle

skeletal: scapule

skeletal: forelimb

skeletal: sternum

skeletal: rib

skeletal: supernumerary rib

skeletal: vertebra

skeletal: pelvic girdle

skeletal: hindlimb

visceral/soft tissue: integumentary

visceral/soft tissue: gastrointestinal tract

visceral/soft tissue: hepatobiliary

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

visceral/soft tissue: heamatopoietic

visceral/soft tissue: immune system

visceral/soft tissue: musculoskeletal system

visceral/soft tissue: nervous system

visceral/soft tissue: central nervous system

visceral/soft tissue: peripheral nervous system

visceral/soft tissue: somatic nervous system

visceral/soft tissue: autonomic nervous system

visceral/soft tissue: endocrine system

visceral/soft tissue: respiratory system

visceral/soft tissue: male reproductive system

visceral/soft tissue: female reproductive system

visceral/soft tissue: eye

visceral/soft tissue: ear

Key result

Developmental effects observed:

no

Conclusions:

In conclusion, the oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of the test item for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.

Executive summary:

The objective of this prenatal developmental toxicity study inSprague Dawley rats conducted as per OECD test guidelines 414,wasto provide general information concerning the effects of prenatal exposure of test item on the pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity in pregnant females and also structural abnormalities or altered growth in the fetuses. The aim ofthis study was to estimate
no-observed-adverse-effect-level (NOAEL) of the test itemfor both maternal as well as fetal end points.

 

A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of111, 333 and 1000mg/kg body weight/dayfor low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

 

Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤15%.

 

A total of 21 (out of 25), 22 (out of 25), 21 (out of 25), and 22 (out of 25) females from group G1, G2, G3 and G4 were found with implantations and live fetuses yielding to pregnancy with rates of 84%, 88%, 84% and 88% respectively. In group G4, 1 out of 25 females was noted with total implantation loss with no evidence of live fetuses.

 

All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on GD 20 by exposing to CO₂asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones such as, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams from each group were evaluated for histopathology of thyroid along with the parathyroid

 

All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.

 

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups. There were no changes noted in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase, maternal feed consumption, number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam. There were no changes noted in mean gravid uterus weight in all the dose levels. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.

 

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

1 Reliable

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

No classification

There are no indicators of reproductive toxicity identified in available studies.