2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.03,19.05,17.06,14.07,12.023,25.024,32.025,30]hexatriaconta-1(36),2,5,7,9,11,14,16,18,20,23,25,27,29,32,34-hexadecaene

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity - fertility - In an oral study according to OECD TG 421 in rats the substance did not exert any reproductive toxic effects (fertility).

Link to relevant study records

Reference

Reference 1

Endpoint:

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

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Pigment Violet 23

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no GLP-Studies available
- Available non-GLP studies: There are no non-GLP-Studies available
- Historical human/control data. There are no human data available
- (Q)SAR: No validated QSAR-models exist for reproductive toxicity endpoints
- In vitro methods: No validated in vitro-models exist for reproductive toxicity endpoints
- Weight of evidence: There is insufficient information available on possible reproductive effects of the registered substance
- Grouping and read-across: There is insufficient information available on possible reproductive effects of the registered substance and structural analogues.
- Substance-tailored exposure driven testing [if applicable]: not applicable
- Approaches in addition to above [if applicable]: not applicable
- Other reasons [if applicable]: not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Specific adaptions of annexes 6 to 10 are not applicable because there are no indications that the substance is mutagenic, carcinogenic or a reproductive toxicant. Though the registered substance as an organic pigment is considered non-reactive, extremely poorly soluble, and hence not bio-available, there are insufficient data available to prove this assumption.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
There is a study according to OECD 421 going on, which will be used to determine the dose range and the final design of the planned study. Results of this OECD 421 study will be available in about 3 months (End of July 2021)

Qualifier:

according to guideline

Guideline:

EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Justification for study design:

The study design is not yet fixed. The final study design will be determined based on the results of an ongoing OECD 421 study.
As there are no indications of reproduction toxicity in available data and the material is considered not-bio-available, the basic design of the study is considered probable.

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 14 days
- Basis for dose level selection: a dose range finding study (OECD 421) is ongoing
- Inclusion/exclusion of extension of Cohort 1B : no extension of cohort 1B
- Termination time for F2: no F2-generation
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: not yet determined
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: not yet determined
- Route of administration: oral gavage

Species:

rat

Sex:

male/female

Route of administration:

oral: gavage

Control animals:

yes, concurrent vehicle

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 February 2021 to 03 August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

As per OECD Guideline No. 421, “Reproduction/Developmental Toxicity Screening Test”, adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN:

- Premating exposure duration for parental (P0) animals: 2 Weeks.

- Basis for dose level selection: The doses of 0, 111, 333 and 1000 mg/kg body weight/day had been selected for vehicle control, low dose, mid dose and high dose groups respectively, as per sponsor’s specification.

- Route of administration: The vehicle and test item formulations were administered through the oral (gavage) route as it is the probable route of human exposure.

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: Rat and Sprague Dawley (standard laboratory rodent species and strain used for toxicity assessment and also recommended by various regulatory authorities.

Propylene glycol was selected as vehicle for test item formulations since the recovery of test item in the prepared formulations was in the acceptance range at a concentration of 200 mg/ml as per in-house trails.

Total Number of Animals: 96 (48 Males + 48 Females)
A total of 48 males and 58 females were received. The additional 10 females recieved for oestrus cycle screening and were sacrificed after grouping and randomization. Females used were nulliparous and non-pregnant.

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)

- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble in distilled water and not suspended uniformly in 0.5% w/v carboxymethyl cellulose at the concentration of 100 mg/mL (considering highest dose of 1000 mg/kg body weight with a dose volume of 10 mL/kg body weight) based on the in-house solubility/suspendibility test results.

The test item was suspended uniformly in corn oil at the concentration of 100 mg/mL (considering highest dose of 1000 mg/kg body weight with a dose volume of 10 mL/kg body weight) and in propylene glycol at the concentration of 200 mg/mL considering highest dose of 1000 mg/kg body weight with a dose volume of 5 mL/kg body weight) as per in-house suspendibility test results.

Based on the above results, propylene glycol was selected as vehicle for test item formulations since the recovery of test item in the prepared formulations was in the acceptance range at a concentration of 200 mg/ml as per in-house trails.

The test item formulations were stable and homogeneous up to 6 hours followed by 48 hours at room temperature at the concentrations of 10 mg/mL and 200 mg/mL in propylene glycol. The average values were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

FORM AS APPLIED IN THE TEST: The test item/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.

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: In-house bred animals.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: 9 to 10 Weeks.
- Weight at study initiation: (P) Males: 232.00 to 285.03 g, Females: 220.05 to 265.13 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 a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- During acclimatization, two animals of the same sex were housed.
- Pre-mating - Per cage, two animals of the same sex and group, were housed.
- Cohabitation Period (mating) - Per cage, two animals (one male and one female) of the same group were housed.
- Post-mating - After confirming the 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.
Note: Sterilized paper shreds were provided as nesting material for main group females from gestation day 20 onwards.
- Diet (ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co.KG)
- Water (ad libitum): Water was available ad libitum throughout the acclimatization and experimental periods. Deep borewell water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimatization period: 5 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5 to 22.8oC
- Humidity (%): 47 to 66%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 04 March 2021 To: 28 May 2021; Experimental End [post-inlife]: 08 July 2021

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared before dose administration and administered within stability period. The required quantity of test item was weighed and triturated well in a mortar with a small quantity of vehicle until a homogenous suspension was formed. After that, the entire quantity of formulation was transferred into a measuring cylinder. A small amount of vehicle was added to rinse the mortar and transferred into the same measuring cylinder. The rinsing procedure of mortar and pestle was repeated to ensure the transfer of the contents to the measuring cylinder. Finally, the volume was made up to required quantity with the vehicle to get desired concentration of 22.2, 66.6 and 200 mg/mL of test item for low, mid and high dose groups, respectively. The test formulations were maintained under stirring conditions using a magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle: The test item was insoluble in distilled water and not suspended uniformly in 0.5% w/v carboxymethyl cellulose at the concentration of 100 mg/mL (considering highest dose of 1000 mg/kg body weight with a dose volume of 10 mL/kg body weight) based on the in-house solubility/suspendibility test results.

The test item was suspended uniformly in corn oil at the concentration of 100 mg/mL (considering highest dose of 1000 mg/kg body weight with a dose volume of 10 mL/kg body weight) and in propylene glycol at the concentration of 200 mg/mL considering highest dose of 1000 mg/kg body weight with a dose volume of 5 mL/kg body weight) as per in-house suspendibility test results.

Based on the above results, propylene glycol was selected as vehicle for test item formulations since the recovery of test item in the prepared formulations was in the acceptance range at a concentration of 200 mg/ml as per in-house trails.

Propylene glycol is accepted vehicle in oral toxicity studies (Reference: Tolerable Levels of Nonclinical Vehicles and Formulations Used in Studies by Multiple Routes in Multiple Species With Notes on Methods to Improve Utility, Shayne Cox Gad et al, International Journal of Toxicology).

- Concentration in vehicle: 0 mg/mL (vehicle control), 22.2 mg/mL (low dose), 66.6 mg/mL (mid dose) and 200 mg/mL (high dose).
- Amount of vehicle (if gavage): 5 mL/kg body weight
- Batch no.: K19A/0319/0711/32
- Make: SD FINE-CHEM LIMITED

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 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No such incidences.
- After successful mating each pregnant female was caged: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually during gestation and lactation periods. Sterilized paper shreds were provided as a nesting material 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 done by the Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 1728. Sampling and analysis of formulations were performed during week 1 and week 4 of the treatment. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers for low, mid and high dose concentrations and in duplicate from a single layer for vehicle control.

The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to the 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 for backup purpose at established stability conditions. The second set of samples were discarded as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, since the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was <10%.

Duration of treatment / exposure:

The males were treated for a period of two weeks (14-days) during pre-mating, during cohabitation period and up to the day before scheduled sacrifice (total of completion of 35 days of treatment).

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 66 days). The non-pregnant females were treated during two-week pre-mating period, during mating until confirmed as mated and further 25 days from the day of confirmation of mating (group G1 - one female until experimental day 51; group G2 - one female until experimental day 52, one female until experimental day 43; G3 - one female until experimental day 51; G4 - one female until experimental day 41).

Frequency of treatment:

Once Daily

Details on study schedule:

- Age at mating of the mated animals in the study: 14 to 15 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

No. of animals per sex per dose:

12 Males + 12 Females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 111, 333 and 1000 mg/kg body weight/day had been selected for vehicle control, low dose, mid dose and high dose groups respectively, as per sponsor’s specification.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once Daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The males were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The 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 fasting/terminal body weights were also recorded before scheduled sacrifice.

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). Only females with normal oestrus cyclicity were selected for treatment. Vaginal smears were monitored daily from the beginning of treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which might induce pseudopregnancy.
The status of oestrus cyclicity of females was also determined on the day of termination.

Sperm parameters (parental animals):

Parameters examined in all parental 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 35 days of treatment.
- Maternal animals: All animals were sacrificed on lactation day 14 and the non-pregnant females were sacrificed after 25 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 [Epididymides, testes, ovaries and vagina] were prepared for microscopic examination and Epididymides, testes, Prostate, Seminal vesicles and coagulating glands, ovaries and uterus with cervix were weighed.

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 data (in the form of appendix) was verified with raw data. After verification, the data was subjected to various statistical analysis using SPSS software version 22.

All analysis and comparisons were evaluated at 95% level of confidence (P<0.05).

Note: Data of non-pregnant females (with positive mating signal) was excluded for mean calculations and statistical analysis for determination of reproductive toxicity end points but considered for systemic toxicity end points. However, the individual animal data is presented the study report.

The statistical analysis was followed but not limited to the parameters in the mentioned below:
The Parametric - One-way ANOVA with Dunnett’s post test was followed to the parameters as
mentioned below:
• Body weight (weekly/gestation/lactation)
• Percent change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Absolute/relative organ weights
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum T4 values
The Non Parametric - Kruskal-Wallis method was followed to the parameters as mentioned below:
• 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
The Cross Tabs - Chi-square test was followed to the parameters as mentioned below:
• Reproductive performances (frequency occurrences)
- mated/non-mated;
- fertile/infertile;
- pregnant/non-pregnant;
- with/without live pups;
- with/without dead pups.

Reproductive indices:

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 confirmed as 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 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 / No. 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 live 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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related clinical signs of toxicity and no mortality/morbidity noted in any of the tested dose group animals of both sexes throughout the experimental period. The noted dark violet coloured faeces from group G4 animals of both sexes from day 2 onwards were due to coloured nature of the test item but not due to adverse changes caused by the test item.

Mortality:

no mortality observed

Description (incidence):

There were no test item-related mortality/morbidity noted in any of the tested dose group animals of both sexes throughout the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 of both sexes during the experimental period in any of the tested dose groups when compared with vehicle control group.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption during of both sexes during premating period in any of the tested dose groups when compared with vehicle control group.
There were no changes noted in mean gestational feed consumption in any of the tested dose groups when compared with vehicle control group.
There were no test-item related changes noted in mean feed consumption during lactation period in any of the tested dose groups when compared with vehicle control group. The noted statistically significant increase in mean lactation feed consumption during lactation day 1 to 4 in group G2 when compared with vehicle control is considered as incidental and toxicologically in-significant.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no histopathological changes noted in any of the organ (ovaries, testes and epididymides with particular emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure) subjected for microscopic examination from vehicle control and high dose groups. Hence, the histopathological examination was not extended to lower dose groups.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Thyroxine (T4) Hormone Levels - Adult Males: There were no changes observed in mean serum Thyroxine (T4) hormone levels at all the tested dose groups (adult males) when compared with vehicle control group. The examination was not extended to adult females as there were no changes noted in serum Thyroxine (T4) hormone levels of adult males.

Serum Thyroxine (T4) Hormone Levels - Pups: There were no changes observed in mean serum Thyroxine (T4) hormone levels of PND 13 pups (pooled samples) representing all the litters from all the tested dose group when compared with vehicle control group. The examination was not extended to PND 4 pups as there were no changes noted in serum Thyroxine (T4) hormone levels of PND 13 pups.However, statistically significant decrease in T4 levels of group G2 and G3 were noted when compared to vehicle control group. This change is considered as incidental as the obtained mean values are within in house historical control data range and also the changes were not occurred in dose dependent manner.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of females from any of the tested dose groups during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during pre-mating and mating treatment period was unaffected by the test item administration in any of the tested dose groups and the mean length was comparable with the vehicle control group. However, one female from group G3 was noted with a single irregular oestrus cycle during pre-mating period in continuation with mating period. This observed irregular cycle is considered as incidental and un-related to treatment as this female was noted with normal cycles further during mating period and also confirmed as pregnant.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Male Mating Index (%): A total of 11 (out of 12), 9 (out of 12), 12 (out of 12) and 10 (out of 12) males were confirmed with mating with a mating index of 91.7%, 75.0%, 100.0% and 83.3% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with vehicle control group.

Male Fertility Index (%): A total of 10 (out of 11), 7 (out of 9), 11 (out of 12) and 9 (out of 10) males were confirmed as impregnating a female with a fertility index of 90.9%, 77.8%, 91.7% and 90.0% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with vehicle control group.

Female Mating Index: All the 12 females from each tested dose groups (G2, G3 and G4) and vehicle control group (G1) were confirmed with mating with a mating index of 100% for each group.

Female Fertility Index (%): A total of 11 (out of 12), 10 (out of 12), 11 (out of 12) and 11 (out of 12) females were confirmed with pregnancy (presence of implantations /presence of live or dead fetuses / evidence of parturition) with a fertility index of 91.7%, 83.3%, 91.7% and 91.7% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for female fertility indices in any of the tested dose groups when compared with vehicle control group.

Pre-coital Interval (Days): A total of 12 pairs were left for cohabitation initially from each tested dose group and vehicle control group. The mean pre-coital interval was 8.08, 10.58, 8.33 and 7.25 days for groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Gestation Length (Days): The mean gestation length [confirmation of mating to parturition] was 22.55, 22.20, 22.45 and 22.27 days for groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Gestation Index / Parturition Index (%): A total of 11 (out of 11), 10 (out of 10), 11 (out of 11), and 11 (out of 11) females were confirmed with live born pups with a gestation / parturition index of 100% from all the groups G1, G2, G3 and G4 respectively.

Pregnancy Index (%): A total of 11 (out of 12), 10 (out of 12), 11 (out of 12) and 11 (out of 12) females were confirmed with pregnancy with a pregnancy index of 91.7%, 83.3%, 91.7% and 91.7% from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Sex Ratio (m/f): Mean sex ratios (m/f) per litter at birth and lactation day 4 were 1.21, 1.26, 1.21 and 1.22 in groups G1, G2, G3 and G4, respectively. Mean sex ratios (m/f) per litter on lactation day 7 and lactation day 13 were 1.50, 1.63, 1.64 and 1.58 in groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Live Birth Index (%) per litter: Mean live birth index (%) per litter were 99.24%, 99.00%, 99.17% and 100.00 % in groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Pup Survival index (%) on LD 4/7/13: Mean pup survival index (%) on LD 4/7/13 per litter was 100% in all groups.

Post implantation loss per litter (No.) and (%): Mean post implantation losses per litter was 0.55, 0.40, 0.27 and 0.27 with a mean percentage of 4.49, 3.37, 2.30 and 2.00 from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dose groups when compared with the vehicle control group.

Mean number of Implantations (No.): The mean number of implantations per litter was 11.45, 11.40, 11.64 and 11.73 from groups G1, G2, G3 and G4, respectively. There were no statistically significant differences noted for this parameter in any of the tested dosed groups when compared with the vehicle control group.

Postnatal losses and (%): There were no postnatal losses noted in any of the litters from all the tested dose groups and vehicle control group.

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

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive performance

other: Serum Thyroxine (T4) Hormone Levels

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no external anomalies noted during daily observation of pups in any of the tested dose groups and vehicle control group litters during postnatal period. All the pups were noted with normal behaviour during daily observations.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no test item-related mortalities noted during daily observation of pups in any of the tested dose groups and vehicle control group litters during postnatal period. All the pups were noted with normal behaviour during daily observations.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related changes observed in mean pup [both male and female] weight per litter recorded on Postnatal Day (PND) 1, 4, 7 and 13 in any of the tested dose group litters when compared with vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross pathological changes observed during necropsy in any of the pups from all the tested dose groups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

gross pathology

other: anogenital distance

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

SUMMARY TABLE

		Group & Dose (mg/kg body weight/day)
Parameters ↓		G1 & 0	G2 & 111	G3 & 333	G4 &1000
Reproductive Indices
No. of Pairs started		12	12	12	12
Reproductive Performance / Indices (males)
Total No. of males cohabited		12	12	12	12
No. of males with confirmed mating		11	9	12	10
No. of males impregnating/siring a female		10	7	11	9
Male Mating Index (%)		91.7	75.0	100.0	83.3
Male Fertility Index (%)		83.3	58.3	91.7	75.0
Reproductive Performance and Indices (females)
Mean Oestrus cycle length (days)	Mean	4.58	4.65	4.49	4.59
	±SD	0.21	0.25	0.30	0.30
No. of females with regular oestrus cycle		12	12	11	11
Total No. of females cohabited		12	12	12	12
No. of sperm-positive females		12	12	12	12
No. of females with evidence of pregnancy		11	10	11	11
No. of females with live born pups		11	10	11	11
No. of females with conceiving days 1 to 5		2	2	3	7
No. of females with conceiving days ≥6		10	10	9	5
Pre-coital Interval (Days)	Mean	8.08	10.58	8.33	7.25
	±SD	4.56	4.83	4.75	6.50
Female Mating Index (%)		100.0	100.0	100.0	100.0
Female Fertility Index (%)		91.7	83.3	91.7	91.7
No. of females with Pregnancy≤21 days		-	-	-	-
No. of females with Pregnancy = 22 days		5	8	6	8
No. of females with Pregnancy≥23 days		6	2	5	3
Gestation Length (Days)	Mean	22.55	22.20	22.45	22.27
	±SD	0.52	0.42	0.52	0.47
Gestation Index (%)		100.0	100.0	100.0	100.0
Female Fecundity or Pregnancy Index (%)		91.7	83.3	91.7	91.7
Parturition index (%)		100.0	100.0	100.0	100.0

SUMMARY TABLE

		Group & Dose (mg/kg body weight/day)
Parameters ↓		G1 & 0	G2 & 111	G3 & 333	G4 &1000
Delivery and Litter Observations
No. of Dams with live young born		11	10	11	11
No. of Dams with live young at PND 4		11	10	11	11
No. of Dams with live young at PND 7		11	10	11	11
No. of Dams with live young at PND 13		11	10	11	11
No. of Implants/dam	Mean	11.45	11.40	11.64	11.73
	±SD	1.13	1.51	1.12	1.62
No. of Live pups/dam at birth	Mean	10.91	11.00	11.36	11.45
	±SD	1.04	1.49	1.12	1.29
No. of Live pups/dam at PND 4	Mean	10.91	11.00	11.36	11.45
	±SD	1.04	1.49	1.12	1.29
No. of Live pups/dam at PND 7	Mean	10.09	10.00	10.18	10.27
	±SD	0.30	0.67	0.40	0.47
No. of Live pups/dam at PND 13	Mean	10.09	10.00	10.18	10.27
	±SD	0.30	0.67	0.40	0.47
Live Birth Index/dam (%)	Mean	99.24	99.00	99.17	100.00
	±SD	2.51	3.16	2.74	0.00
Pup Survival Index/dam on PND 4 (%)	Mean	100.00	100.00	100.00	100.00
	±SD	0.00	0.00	0.00	0.00
Pup Survival Index/dam on PND 7 (%)	Mean	100.00	100.00	100.00	100.00
	±SD	0.00	0.00	0.00	0.00
Pup Survival Index/dam on PND 13 (%)	Mean	100.00	100.00	100.00	100.00
	±SD	0.00	0.00	0.00	0.00
Sex ratio (m/f)/dam At Birth	Mean	1.21	1.26	1.21	1.22
	±SD	0.56	0.24	0.35	0.45
Sex ratio (m/f)/dam at PND 4	Mean	1.21	1.26	1.21	1.22
	±SD	0.56	0.24	0.35	0.45
Sex ratio (m/f)/dam at PND 7	Mean	1.50	1.63	1.64	1.58
	±SD	0.92	0.57	0.67	0.58
Sex ratio (m/f)/dam at PND 13	Mean	1.50	1.63	1.64	1.58
	±SD	0.92	0.57	0.67	0.58

SUMMARY TABLE

		Group & Dose (mg/kg body weight/day)
Parameters ↓		G1 & 0	G2 & 111	G3 & 333	G4 &1000
Delivery and Litter Observations
No. of Dams with live young born		11	10	11	11
No. of Dams with live young at PND 4		11	10	11	11
No. of Dams with live young at PND 7		11	10	11	11
No. of Dams with live young at PND 13		11	10	11	11
No. of Implants/dam	Mean	11.45	11.40	11.64	11.73
	±SD	1.13	1.51	1.12	1.62
No. of Live pups/dam at birth	Mean	10.91	11.00	11.36	11.45
	±SD	1.04	1.49	1.12	1.29
No. of Live pups/dam at PND 4	Mean	10.91	11.00	11.36	11.45
	±SD	1.04	1.49	1.12	1.29
No. of Live pups/dam at PND 7	Mean	10.09	10.00	10.18	10.27
	±SD	0.30	0.67	0.40	0.47
No. of Live pups/dam at PND 13	Mean	10.09	10.00	10.18	10.27
	±SD	0.30	0.67	0.40	0.47
Live Birth Index/dam (%)	Mean	99.24	99.00	99.17	100.00
	±SD	2.51	3.16	2.74	0.00
Pup Survival Index/dam on PND 4 (%)	Mean	100.00	100.00	100.00	100.00
	±SD	0.00	0.00	0.00	0.00
Pup Survival Index/dam on PND 7 (%)	Mean	100.00	100.00	100.00	100.00
	±SD	0.00	0.00	0.00	0.00
Pup Survival Index/dam on PND 13 (%)	Mean	100.00	100.00	100.00	100.00
	±SD	0.00	0.00	0.00	0.00
Sex ratio (m/f)/dam At Birth	Mean	1.21	1.26	1.21	1.22
	±SD	0.56	0.24	0.35	0.45
Sex ratio (m/f)/dam at PND 4	Mean	1.21	1.26	1.21	1.22
	±SD	0.56	0.24	0.35	0.45
Sex ratio (m/f)/dam at PND 7	Mean	1.50	1.63	1.64	1.58
	±SD	0.92	0.57	0.67	0.58
Sex ratio (m/f)/dam at PND 13	Mean	1.50	1.63	1.64	1.58
	±SD	0.92	0.57	0.67	0.58

SUMMARY TABLE

			Group & Dose (mg/kg body weight/day)
Parameters ↓			G1 & 0	G2 & 111	G3 & 333	G4 &1000
Delivery and Litter Observations
Mean Male Pup weight (g)/dam	At birth	Mean	6.42	6.43	6.45	6.41
		±SD	0.17	0.22	0.19	0.10
	PND 4	Mean	10.85	10.78	10.95	10.76
		±SD	0.27	0.31	0.29	0.26
	PND 7	Mean	15.64	15.58	15.78	15.56
		±SD	0.18	0.17	0.23	0.60
	PND 13	Mean	25.52	25.49	25.50	25.50
		±SD	0.17	0.21	0.30	0.26
Mean Female Pup weight (g)/dam	At Birth	Mean	5.71	5.70	5.71	5.76
		±SD	0.15	0.12	0.15	0.14
	PND 4	Mean	9.66	9.82	10.01	10.01
		±SD	0.14	0.31	0.40	0.39
	PND 7	Mean	13.90	14.12	14.48	14.13
		±SD	0.21	0.38	0.55	0.63
	PND 13	Mean	24.03	24.07	24.23	24.05
		±SD	0.34	0.40	0.38	0.65
Mean Male Pup AGD Measurement/dam (mm)on PND 4		Mean	4.44	4.36	4.39	4.42
		±SD	0.17	0.13	0.15	0.12
Mean Male Pup AGD ratio/dam on PND 4		Mean	2.01	1.98	1.98	2.00
		±SD	0.09	0.07	0.08	0.06
Mean Female Pup AGD Measurement/dam (mm)on PND 4		Mean	2.50	2.45	2.44	2.46
		±SD	0.10	0.13	0.09	0.10
Mean Female Pup AGD ratio/dam on PND 4		Mean	1.17	1.15	1.13	1.14
		±SD	0.05	0.07	0.04	0.06
Male Pup Nipple Retention/dam on PND 13		Mean	0.00	0.00	0.00	0.00
		±SD	0.00	0.00	0.00	0.00

SUMMARY TABLE

		Group & Dose (mg/kg body weight/day)
Parameters ↓		G1 & 0	G2 & 111	G3 & 333	G4 &1000
ABNORMAL PUPS
No. of Dams with 0 abnormal pups		11	10	11	11
LOSS OF OFFSPRING
Pre-natal or post-implantation loss (implantations minus live births)
No. of Females with 0 post-implantation loss		7	7	8	8
No. of Females with 1 post-implantation loss		2	2	3	3
No. of Females with 2 post-implantation losses		2	1	-	-
Post-implantation Loss (No.)	Mean	0.55	0.40	0.27	0.27
	±SD	0.82	0.70	0.47	0.47
Post-implantation Loss (%)	Mean	4.49	3.37	2.30	2.00
	±SD	6.80	5.70	3.97	3.42
Post-natal loss (live births minus alive at PND 13)
No. of Females with 0
Post-natal loss(No.)	Mean	0.00	0.00	0.00	0.00
	±SD	0.00	0.00	0.00	0.00
Post-natal Loss (%)	Mean	0.00	0.00	0.00	0.00
	±SD	0.00	0.00	0.00	0.00

TABLE 1.      SUMMARY OF CLINICAL SIGNS OF TOXICITY 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)	Mortality^b: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	Day 1 to termination:N (12)	0 (12)
G2, M & 111	12	Day 1 to termination:N (12)	0 (12)
G3, M & 333	12	Day 1 to termination:N (12)	0 (12)
G4, M & 1000	12	Day 1 to termination:N^@(12)	0 (12)

M: Male; N: Normal. N^@:Noted with dark violet coloured faeces from group G4 animals from day 2 onwards. This coloration is due to coloured nature of the test item but not due adverse changes caused by the test item
a: observed daily once; b: observed twice daily.

TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS OF TOXICITY 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)	Mortality^b No. of Mortalities (Total No. of Animals)
G1, F & 0	12	Day 1 to termination:N (12)	0 (12)
G2, F & 111	12	Day 1 to termination:N (12)	0 (12)
G3, F & 333	12	Day 1 to termination:N (12)	0 (12)
G4, F & 1000	12	Day 1 to termination:N^@(12)	0 (12)

F: Female; N: Normal; N^@:Noted with dark violet coloured faeces from group G4 animals from day 2 onwards. This coloration is due to coloured nature of the test item but not due adverse changes caused by the test item.
a: observed daily once; b: observed twice daily.

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	35
G1, M & 0	Mean	327.23	342.10	355.42	367.81	381.79	400.60
	±SD	21.41	24.14	27.32	33.87	38.41	37.85
	n	12	12	12	12	12	12
G2, M & 111	Mean	328.87	340.76	354.88	370.15	388.65	405.46
	±SD	23.43	24.85	27.78	31.70	25.99	32.40
	n	12	12	12	12	12	12
G3, M & 333	Mean	328.49	341.83	356.47	366.08	384.78	402.24
	±SD	22.47	27.14	32.71	37.53	45.21	41.33
	n	12	12	12	12	12	12
G4, M & 1000	Mean	329.76	338.18	361.99	371.91	390.03	409.56
	±SD	21.61	26.04	31.64	35.57	34.89	32.27
	n	12	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^#	28^#
G1, F & 0	Mean	258.31	264.36	270.49	272.88	270.32
	±SD	13.50	15.70	16.50	7.69	-
	n	12	12	12	6	1
G2, F & 111	Mean	261.58	267.88	276.57	282.11	302.21
	±SD	17.08	22.31	22.74	20.09	23.73
	n	12	12	12	9	3
G3, F & 333	Mean	260.65	265.99	274.35	282.88	-
	±SD	14.38	17.03	18.02	19.14	-
	n	12	12	12	8	-
G4, F & 1000	Mean	259.81	264.56	268.52	264.54	271.37
	±SD	14.34	16.37	19.14	7.33	11.30
	n	12	12	12	4	2

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 and 28 body weights were not subjected to statistical analysis due to uneven number of variables, but presented in individual animal data.

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

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	7	14	20
G1, F & 0	Mean	276.67	288.18	310.69	369.38
	±SD	16.09	15.69	16.38	18.95
	n	11	11	11	11
G2, F & 111	Mean	282.77	292.48	316.43	374.14
	±SD	21.73	22.81	21.65	19.15
	n	10	10	10	10
G3, F & 333	Mean	282.59	292.85	317.90	375.27
	±SD	19.07	17.36	17.91	17.55
	n	11	11	11	11
G4, F & 1000	Mean	272.83	284.39	309.40	368.59
	±SD	16.83	16.75	17.95	21.80
	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 OF LACTATION BODY WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Lactation Day (LD)
		1	4	7	13
G1, F & 0	Mean	292.08	296.88	310.77	324.88
	±SD	16.99	17.20	16.56	14.91
	n	11	11	11	11
G2, F & 111	Mean	294.60	299.52	311.10	326.67
	±SD	20.35	22.07	21.42	19.84
	n	10	10	10	10
G3, F & 333	Mean	294.74	299.67	313.29	326.99
	±SD	19.02	19.03	19.17	17.30
	n	11	11	11	11
G4, F & 1000	Mean	285.52	288.94	302.13	317.03
	±SD	11.55	13.66	14.74	14.31
	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 (%) Gain during Day
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 35
G1, M & 0	Mean	4.53	8.59	12.30	16.54	22.37
	±SD	1.98	3.60	5.21	6.52	7.21
	n	12	12	12	12	12
G2, M & 111	Mean	3.64	7.92	12.54	18.35	23.38
	±SD	2.65	3.55	4.51	5.94	6.72
	n	12	12	12	12	12
G3, M & 333	Mean	4.01	8.40	11.28	16.95	22.32
	±SD	1.95	3.64	5.34	8.24	6.84
	n	12	12	12	12	12
G4, M & 1000	Mean	2.49	9.65	12.62	18.17	24.15
	±SD	1.98	3.65	4.92	5.27	4.54
	n	12	12	12	12	12

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

TABLE 5 (Contd…). 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 (%) Gain during Day
		1 to 7	1 to 14	1 to 21^#	1 to 28^#
G1, F & 0	Mean	2.32	4.69	6.58	11.35
	±SD	1.40	1.96	1.82	-
	n	12	12	6	1
G2, F & 111	Mean	2.32	5.65	8.25	9.79
	±SD	2.38	2.84	2.19	2.14
	n	12	12	9	3
G3, F & 333	Mean	2.01	5.23	7.34	-
	±SD	1.51	2.78	3.37	-
	n	12	12	8	-
G4, F & 1000	Mean	1.80	3.32	3.85	7.37
	±SD	1.07	3.46	2.55	3.14
	n	12	12	4	2

F: Female; SD: Standard Deviation; n: Number of Animals; -: Not applicable.

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

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

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	4.19	7.82	18.94
	±SD	0.96	1.43	3.45
	n	11	11	11
G2, F & 111	Mean	3.43	8.26	18.39
	±SD	1.09	1.63	3.68
	n	10	10	10
G3, F & 333	Mean	3.68	8.58	18.12
	±SD	1.10	1.40	2.72
	n	11	11	11
G4, F & 1000	Mean	4.25	8.80	19.15
	±SD	0.88	1.65	2.95
	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 OF PERCENT CHANGE IN LACTATION BODY WEIGHT GAIN (%) RECORD

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	1.65	4.71	4.58
	±SD	1.11	0.89	1.39
	n	11	11	11
G2, F & 111	Mean	1.65	3.91	5.07
	±SD	1.39	1.63	1.77
	n	10	10	10
G3, F & 333	Mean	1.68	4.56	4.42
	±SD	1.07	0.88	1.04
	n	11	11	11
G4, F & 1000	Mean	1.18	4.56	4.95
	±SD	1.18	1.05	0.95
	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	22.30	24.10
	±SD	1.73	1.16
	n	12 (6)	12 (6)
G2, M & 111	Mean	22.39	23.53
	±SD	1.94	1.33
	n	12 (6)	12 (6)
G3, M & 333	Mean	21.90	23.30
	±SD	1.86	1.40
	n	12 (6)	12 (6)
G4, M & 1000	Mean	21.29	23.49
	±SD	1.45	0.88
	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	15.95	17.92
	±SD	0.78	0.57
	n	12 (6)	12 (6)
G2, F & 111	Mean	17.13	18.38
	±SD	0.86	1.11
	n	12 (6)	12 (6)
G3, F & 333	Mean	16.62	17.65
	±SD	1.17	1.08
	n	12 (6)	12 (6)
G4, F & 1000	Mean	15.16	17.45
	±SD	0.80	1.63
	n	12 (6)	12 (6)

F: Female; 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	19.03	21.14	25.16
	±SD	0.76	1.17	1.17
	n	11	11	11
G2, F & 111	Mean	19.37	21.79	26.69
	±SD	1.37	1.56	1.95
	n	10	10	10
G3, F & 333	Mean	18.83	21.47	25.62
	±SD	1.60	1.31	1.42
	n	11	11	11
G4, F & 1000	Mean	18.82	20.99	25.04
	±SD	1.69	1.73	1.95
	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	27.15	30.44	34.40
	±SD	1.36	1.84	1.14
	n	11	11	11
G2, F & 111	Mean	29.01*	32.01	35.62
	±SD	1.80	2.59	1.74
	n	10	10	10
G3, F & 333	Mean	27.81	30.91	34.89
	±SD	1.52	2.22	1.83
	n	11	11	11
G4, F & 1000	Mean	27.24	30.45	34.30
	±SD	1.89	2.45	2.13
	n	11	11	11

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

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

TABLE 11.      SUMMARY RECORD OF VAGINAL SMEAR EXAMINATION FOR DETERMINATION OF OESTRUS CYCLICITY

Determination of Oestrus Cyclicity during Pre-Mating Treatment Period
Group, Sex & Dose (mg/kg body weight/day)	Total No. of Females Evaluated		No. of Females with Complete Regular Oestrus Cycle	No. of Females with at least one Irregular Oestrus Cycle	Average Length of Oestrus Cycle (Days)
G1, F & 0	12	n	12	0	Mean	4.58
		%	100.0	0.0	±SD	0.21
					n	12
G2, F & 111	12	n	12	0	Mean	4.65
		%	100.0	0.0	±SD	0.25
					n	12
G3, F & 333	12	n	11	1	Mean	4.49
		%	91.7	8.3	±SD	0.30
					n	12
G4, F & 1000	12	n	12	0	Mean	4.61
		%	100.0	0.0	±SD	0.28
					n	12

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

TABLE 12. SUMMARY OF DELIVERY DATA (AT BIRTH) RECORD PER LITTER

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	11.00		5.73	5.18	10.91		0.09	0.00	0.09		0.00	0.00	0.00	0.00		1.21		99.24
	±SD	1.10		1.01	1.25	1.04		0.30	0.00	0.30		0.00	0.00	0.00	0.00		0.56		2.51
	n	11		11	11	11		11	11	11		11	11	11	11		11		11
G2, F & 111	Mean	11.10		6.10	4.90	11.00		0.00	0.10	0.10		0.00	0.00	0.00	0.00		1.26		99.00
	±SD	1.37		1.10	0.74	1.49		0.00	0.32	0.32		0.00	0.00	0.00	0.00		0.24		3.16
	n	10		10	10	10		10	10	10		10	10	10	10		10		10
G3, F & 333	Mean	11.45		6.09	5.27	11.36		0.00	0.09	0.09		0.00	0.00	0.00	0.00		1.21		99.17
	±SD	1.04		1.04	1.10	1.12		0.00	0.30	0.30		0.00	0.00	0.00	0.00		0.35		2.74
	n	11		11	11	11		11	11	11		11	11	11	11		11		11
G4, F & 1000	Mean	11.45		6.09	5.36	11.45		0.00	0.00	0.00		0.00	0.00	0.00	0.00		1.22		100.00
	±SD	1.29		0.94	1.29	1.29		0.00	0.00	0.00		0.00	0.00	0.00	0.00		0.45		0.00
	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.

TABLE 13.      SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

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	5.73	5.18	10.91		0.00	0.00	0.00		0.00	0.00	0.00		1.21		100.00
	±SD	1.01	1.25	1.04		0.00	0.00	0.00		0.00	0.00	0.00		0.56		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G2, F & 111	Mean	6.10	4.90	11.00		0.00	0.00	0.00		0.00	0.00	0.00		1.26		100.00
	±SD	1.10	0.74	1.49		0.00	0.00	0.00		0.00	0.00	0.00		0.24		0.00
	n	10	10	10		10	10	10		10	10	10		10		10
G3, F & 333	Mean	6.09	5.27	11.36		0.00	0.00	0.00		0.00	0.00	0.00		1.21		100.00
	±SD	1.04	1.10	1.12		0.00	0.00	0.00		0.00	0.00	0.00		0.35		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G4, F & 1000	Mean	6.09	5.36	11.45		0.00	0.00	0.00		0.00	0.00	0.00		1.22		100.00
	±SD	0.94	1.29	1.29		0.00	0.00	0.00		0.00	0.00	0.00		0.45		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; LD: Lactation Day.

TABLE 13 (Contd…). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		Pups Sacrificed for Hormonal analysis (No.)				Live Pups (No.) on LD 4 after 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	0.82	0.82		5.73	4.36	10.09		5.73	4.36	10.09		0.00	0.00	0.00		0.00	0.00	0.00		1.50		100.00
	±SD	0.00	0.87	0.87		1.01	1.03	0.30		1.01	1.03	0.30		0.00	0.00	0.00		0.00	0.00	0.00		0.92		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	1.00	1.00		6.10	3.90	10.00		6.10	3.90	10.00		0.00	0.00	0.00		0.00	0.00	0.00		1.63		100.00
	±SD	0.00	0.94	0.94		1.10	0.57	0.67		1.10	0.57	0.67		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	10		10	10	10		10		10
G3, F & 333	Mean	0.00	1.18	1.18		6.09	4.09	10.18		6.09	4.09	10.18		0.00	0.00	0.00		0.00	0.00	0.00		1.64		100.00
	±SD	0.00	0.87	0.87		1.04	1.04	0.40		1.04	1.04	0.40		0.00	0.00	0.00		0.00	0.00	0.00		0.67		0.00
	n	11	11	11		11	11	11		11	11	11		11	11	11		11	11	11		11		11
G4, F & 1000	Mean	0.00	1.18	1.18		6.09	4.18	10.27		6.09	4.18	10.27		0.00	0.00	0.00		0.00	0.00	0.00		1.58		100.00
	±SD	0.00	0.98	0.98		0.94	0.98	0.47		0.94	0.98	0.47		0.00	0.00	0.00		0.00	0.00	0.00		0.58		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 13 (Contd…). SUMMARY OF LITTER OBSERVATION RECORD DURING LACTATION PERIOD

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	5.73	4.36	10.09		0.00	0.00	0.00		0.00	0.00	0.00		1.50		100.00
	±SD	1.01	1.03	0.30		0.00	0.00	0.00		0.00	0.00	0.00		0.92		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G2, F & 111	Mean	6.10	3.90	10.00		0.00	0.00	0.00		0.00	0.00	0.00		1.63		100.00
	±SD	1.10	0.57	0.67		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	6.09	4.09	10.18		0.00	0.00	0.00		0.00	0.00	0.00		1.64		100.00
	±SD	1.04	1.04	0.40		0.00	0.00	0.00		0.00	0.00	0.00		0.67		0.00
	n	11	11	11		11	11	11		11	11	11		11		11
G4, F & 1000	Mean	6.09	4.18	10.27		0.00	0.00	0.00		0.00	0.00	0.00		1.58		100.00
	±SD	0.94	0.98	0.47		0.00	0.00	0.00		0.00	0.00	0.00		0.58		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.

TABLE 14.      SUMMARY OF REPRODUCTIVE PERFORMANCE RECORD

Group, Sex & Dose (mg/kg body weight/day)	No. of Males with Evidence of Mating (Total 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	11 (12)	91.7		10 (11)	90.9
G2, M & 111	9 (12)	75.0		7 (9)	77.8
G3, M & 333	12 (12)	100.0		11 (12)	91.7
G4, M & 1000	10 (12)	83.3		9 (10)	90.0

M: Male.

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE RECORD

Group, Sex & Dose (mg/kg body weight/day)		Pre-coital Interval / Mean Time to Mating (Days)				Gestation Length / Duration of Pregnancy (Days)
		Pre-Coital Interval / Copulatory Interval		Conceiving Days (1 to 5)	Conceiving Days (5 to More)	Gestation Length (Days)
G1, F & 0	Mean	8.08	n	2	10	22.55
	±SD	4.56	%	16.7	83.3	0.52
	n	12				11
G2, F & 111	Mean	10.58	n	2	10	22.20
	±SD	4.83	%	16.7	83.3	0.42
	n	12				10
G3, F & 333	Mean	8.33	n	3	7	22.45
	±SD	4.75	%	25.0	58.3	0.52
	n	12				11
G4, F & 1000	Mean	7.25	n	7	5	22.27
	±SD	6.50	%	58.3	41.7	0.47
	n	12				11

F: Female; SD: Standard Deviation.

n: Number of Females confirmed with mating (in case of Pre-Coital Interval) / Number of Females confirmed with pregnancy (in case of gestation length).

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE RECORD

Group, Sex & Dose (mg/kg body weight/day)	Female Mating Index (%)				Female Fertility Index (%)				Female Fecundity or Pregnancy 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 (%)		No. of Pregnant Females	No. of Females with confirmed Mating	Female Fecundity or Pregnancy Index (%)
G1, F & 0	12	12	100.0		11	12	91.7		11	12	91.7
G2, F & 111	12	12	100.0		10	12	83.3		10	12	83.3
G3, F & 333	12	12	100.0		11	12	91.7		11	12	91.7
G4, F & 1000	12	12	100.0		11	12	91.7		11	12	91.7

F: Female.

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE RECORD

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

F: Female.

TABLE 14 (Contd…). SUMMARY OF REPRODUCTIVE PERFORMANCE RECORD

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	11.45	10.91	0.55	4.49		0.00	0.00
	±SD	1.13	1.04	0.82	6.80		0.00	0.00
	n	11	11	11	11		11	11
G2, F & 111	Mean	11.40	11.00	0.40	3.37		0.00	0.00
	±SD	1.51	1.49	0.70	5.70		0.00	0.00
	n	10	10	10	10		10	10
G3, F & 333	Mean	11.64	11.36	0.27	2.30		0.00	0.00
	±SD	1.12	1.12	0.47	3.97		0.00	0.00
	n	11	11	11	11		11	11
G4, F & 1000	Mean	11.73	11.45	0.27	2.00		0.00	0.00
	±SD	1.62	1.29	0.47	3.42		0.00	0.00
	n	11	11	11	11		11	11

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

TABLE 15.      SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Testes	Epididymides	Prostate	Seminal vesicles with coagulating glands	Thyroid along with parathyroid^#
G1, M & 0	Mean	3.3720	1.3413	1.4803	1.3816	0.0279
	±SD	0.3374	0.0984	0.3297	0.4225	0.0031
	n	12	12	12	12	12
G2, M & 111	Mean	3.5378	1.3841	1.2655	1.5133	0.0266
	±SD	0.4152	0.0797	0.4030	0.4509	0.0027
	n	12	12	12	12	12
G3, M & 333	Mean	3.4076	1.3575	1.2593	1.7477	0.0271
	±SD	0.3356	0.1761	0.3783	0.4599	0.0036
	n	12	12	12	12	12
G4, M & 1000	Mean	3.3325	1.3280	1.4099	1.7572	0.0268
	±SD	0.3050	0.1226	0.4141	0.3897	0.0036
	n	12	12	12	12	12

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

TABLE 15 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHT (g) RECORD

Group, Sex & Dose (mg/kg body weight/day)		Ovaries	Uterus with cervix	Thyroid along with parathyroid^#
G1, F & 0	Mean	0.1403	0.5631	0.0241
	±SD	0.0132	0.0593	0.0018
	n	12	12	12
G2, F & 111	Mean	0.1434	0.5950	0.0231
	±SD	0.0167	0.0929	0.0014
	n	12	12	12
G3, F & 333	Mean	0.1394	0.5862	0.0242
	±SD	0.0114	0.0930	0.0010
	n	12	12	12
G4, F & 1000	Mean	0.1598*	0.5946	0.0249
	±SD	0.0220	0.1091	0.0014
	n	12	12	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 16.      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)	Testes	Epididymides	Prostate	Seminal vesicles with coagulating glands	Thyroid along with parathyroid
G1, M & 0	Mean	380.22	0.8891	0.3559	0.3900	0.3627	0.0073
	±SD	37.04	0.0711	0.0443	0.0797	0.1035	0.0004
	n	12	12	12	12	12	12
G2, M & 111	Mean	386.90	0.9163	0.3590	0.3268	0.3909	0.0069
	±SD	32.32	0.0978	0.0222	0.0981	0.1087	0.0004
	n	12	12	12	12	12	12
G3, M & 333	Mean	378.99	0.9028	0.3587	0.3328	0.4612	0.0071
	±SD	41.91	0.0752	0.0331	0.0933	0.1183	0.0004
	n	12	12	12	12	12	12
G4, M & 1000	Mean	388.02	0.8635	0.3438	0.3605	0.4584	0.0069
	±SD	32.33	0.0980	0.0361	0.0934	0.1226	0.0008
	n	12	12	12	12	12	12

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

TABLE 16 (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)	Ovaries	Uterus with cervix	Thyroid along with parathyroid
G1, F & 0	Mean	316.95	0.0444	0.1779	0.0076
	±SD	19.26	0.0048	0.0184	0.0005
	n	12	12	12	12
G2, F & 111	Mean	321.31	0.0449	0.1853	0.0072
	±SD	23.76	0.0065	0.0252	0.0007
	n	12	12	12	12
G3, F & 333	Mean	316.28	0.0441	0.1853	0.0077
	±SD	16.77	0.0035	0.0275	0.0005
	n	12	12	12	12
G4, F & 1000	Mean	309.50	0.0521*	0.1922	0.0081
	±SD	17.40	0.0096	0.0333	0.0003
	n	12	12	12	12

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

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

TABLE 17.      SUMMARY RECORD OF SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD -MALES

Group, Sex & Dose (mg/kg body weight/day)		Serum T4 Levels (ng/mL)
G1, M & 0	Mean	64.014
	±SD	5.727
	n	12
G2, M & 111	Mean	70.924
	±SD	7.574
	n	12
G3, M & 333	Mean	66.404
	±SD	8.246
	n	12
G4, M & 1000	Mean	69.331
	±SD	10.651
	n	12

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

TABLE 18.      SUMMARY OF PUP OBSERVATIONS RECORD 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 / Litters^#		11		11		6		11		11
		No. of Live Pups		120		120		9		111		111
		Pup Observation/ No. of Pups observed		N/120		N/120		-		N/111		N/111
G2, F & 111		No. of Dams / Litters^#		10		10		6		10		10
		No. of Live Pups		110		110		10		100		100
		Pup Observation/ No. of Pups observed		N/110		N/110		-		N/100		N/100
G3, F & 333		No. of Dams / Litters^#		11		11		8		11		11
		No. of Live Pups		125		125		13		112		112
		Pup Observation/ No. of Pups observed		N/125		N/125		-		N/112		N/112
G4, F & 1000		No. of Dams / Litters^#		11		11		7		11		11
		No. of Live Pups		126		126		13		113		113
		Pup Observation/ No. of Pups observed		N/126		N/126		-		N/113		N/113

F: Female; N: Normal; PND: Postnatal Day; #: confirmed with live pups.

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

TABLE 19.      SUMMARY OF MEAN PUP WEIGHT (g) PER LITTER RECORD DURING LACTATION PERIOD

Group, Sex & Dose (mg/kg body weight/day)		PND 1			PND 4			PND 7			PND 14
		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	6.42	5.71		10.85	9.66		15.64	13.90		25.52	24.03
	±SD	0.17	0.15		0.27	0.14		0.18	0.21		0.17	0.34
	n	11	11		11	11		11	11		11	11
G2, F & 111	Mean	6.43	5.70		10.78	9.82		15.58	14.12		25.49	24.07
	±SD	0.22	0.12		0.31	0.31		0.17	0.38		0.21	0.40
	n	10	10		10	10		10	10		10	10
G3, F & 333	Mean	6.45	5.71		10.95	10.01*		15.78	14.48*		25.50	24.23
	±SD	0.19	0.15		0.29	0.40		0.23	0.55		0.30	0.38
	n	11	11		11	11		11	11		11	11
G4, F & 1000	Mean	6.41	5.76		10.76	10.01*		15.56	14.13		25.50	24.05
	±SD	0.10	0.14		0.26	0.39		0.60	0.63		0.26	0.65
	n	11	11		11	11		11	11		11	11

F: Female; SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day.

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

TABLE 20.      SUMMARY OF MEAN PUP ANOGENITAL DISTANCE (AGD) MEASUREMENT (mm) AND ANOGENITAL DISTANCE (AGD) RATIO PER LITTER RECORD ON POSTNATAL DAY 4

Group & Dose (mg/kg body weight/day)		Mean Anogenital Distance Measurement (mm)			Mean Anogenital Distance Ratio
		Male	Female		Male	Female
G1 & 0	Mean	4.44	2.50		2.01	1.17
	±SD	0.17	0.10		0.09	0.05
	n	11	11		11	11
G2 & 111	Mean	4.36	2.45		1.98	1.15
	±SD	0.13	0.13		0.07	0.07
	n	10	10		10	10
G3 & 333	Mean	4.39	2.44		1.98	1.13
	±SD	0.15	0.09		0.08	0.04
	n	11	11		11	11
G4 & 1000	Mean	4.42	2.46		2.00	1.14
	±SD	0.12	0.10		0.06	0.06
	n	11	11		11	11

F: Female; SD: Standard Deviation; n: Number of Litters; AGD: Anogenital Distance.

TABLE 21.      SUMMARY OF MALE PUP NIPPLE/AREOLAE RETENTION (no.) RECORD PER LITTER

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
G2 & 111	Mean	0.00
	±SD	0.00
	n	10
G3 & 333	Mean	0.00
	±SD	0.00
	n	11
G4 & 1000	Mean	0.00
	±SD	0.00
	n	11

SD: Standard Deviation; n: Number of Litters.

TABLE 22.      SUMMARY OF SERUM THYROXINE (T4) HORMONE LEVELS (ng/mL) RECORD - POSTNATAL DAY 13 PUPS (PND)

Group & Dose (mg/kg body weight/day)		Serum T4 Levels_Adj. Result (ng/mL)
G1 & 0	Mean	75.981
	±SD	8.229
	n	11
G2 & 111	Mean	65.396*
	±SD	6.096
	n	10
G3 & 333	Mean	66.267*
	±SD	4.283
	n	11
G4 & 1000	Mean	68.517
	±SD	11.201
	n	11

SD: Standard Deviation; n: Number of Litters; PND: Postnatal Day.

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

Conclusions:

In conclusion, the oral administration of the test item 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/day for systemic reproduction and developmental toxicity endpoints.

Executive summary:

The objective of this Reproduction/Developmental Toxicity Screening Testin Sprague Dawleyrats was to determine the possible health hazards likely toarise from repeated exposure of the test item over arelatively limited time. It also provided initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 96 (48 males + 48 females) Sprague Dawley rats were selected for the study and distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 12 rats per sex. G1 animals were administered with vehicle alone [Propylene Glycol]. The G2, G3 and G4 animals were treated with test item at 111, 333 and 1000 mg/kg bw/day, respectively. Vehicle and test item formulations were administered by oral gavage at the fixed dose volume of 5 mL/kg body weight. Male rats were treated before mating, during mating and thereafter for a total of 35 days. Female rats were treated before mating, during mating, during gestation and up to lactation day 13, for a total minimum period of 50 days and a maximum period of 66 days.

Stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item formulations were stable and homogeneous up to 6 hours followed by 48 hours at room temperatureat the concentrations of 10 mg/mL and 200 mg/mL inpropylene glycol.Homogeneity and dose formulation analysis for concentration verification was performed during week 1 and 4 of administration period. The average values 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 animals were observed once daily for clinical signs and twice daily for mortality and morbidity. Body weights were recorded for all animals once weekly and feed consumption was measured coinciding with body weight except during cohabitation. Serum thyroxine hormone (T4) levels were determined in males by ELISA method. All animals were necropsied and examined for gross pathological findings and changes in organ weight. Detailed histopathological examinations of the ovaries, testes and epididymides were performed in G1 and G4 animals, with special emphasis on the stages of spermatogenesis and the interstitial testicular cell structure.

Reproductive performance was determined for both sexes. Male rats were examined for changes in mating and fertility indices. Female rats were examined for changes in mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length. Female rats were also examined for oestrus cyclicity during treatment period until evidence of mating. Gestational body weights were recorded on gestation day (GD) 0, 7, 14 and 20. Lactating body weights were recorded on lactation day (LD) 1, 4, 7 and 13. Terminal body weights were recorded for all animals on the day of sacrifice. Delivery and litter observations such as the number of live/dead pups born, litter size, sex ratio, live birth index and pup viability were noted or calculated in each litter. The numbers of implantation, post-implantation and postnatal losses were recorded.

Pups were observed once daily for external behavioural changes and twice daily for mortality up to the day of sacrifice on postnatal day (PND) 13. Individual pup weights were recorded on PND 1, 4, 7 and 13. Anogenital distance (AGD) measurement to calculate AGD ratio was recorded on PND 4 for each pup. Male pups were examined for nipple/areolae retention on PND 13. All pups were observed for gross pathological findings on PND 13. Serum thyroxine hormone (T4) levels were estimated for PND 13 pups.

All animals survived to scheduled sacrifice and there were no test item-related clinical signs of toxicity or mortalities noted in any of the dose levels. No test item-related changes were noted in body weight or body weight gain and feed consumption of both sexes from any of the dose levels during the experimental period. There were no significant changes noted in serum thyroxine hormone (T4) levels in all the dose groups when compared to the vehicle control. No significant changes in absolute or relative organ weights of both sexes in any of the dose levels were observed. There were no gross pathological changes observed during necropsy in all of the adult animals.

No significant changes in mating or fertility were observed in any of the dose levels of both sexes. Gestation index, parturition index, pre-coital interval and gestation length were unaffected by the treatment in all the tested dose levels. No significant changes in oestrus cyclicity or length were observed in all the tested dose levels. Delivery parameters were unaffected by the treatment, as were litter observations during lactation. No significant changes in the numbers of implantations and post-implantation loss. No postnatal losses were observed at all the tested dose levels. 

All pups were normal externally and there were no treatment-related deaths noted in all the tested dose levels. No significant changes in mean pup weight or AGD measurement/ratio were observed in all the tested dose levels. No cases of nipple retention were observed on PND 13 among male pups. Serum thyroxine (T4) hormone levels measured on PND 13 were unaffected by treatment. No remarkable effects on sex ratio were observed.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable without restrictions

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

Reproductive toxicity - developmental toxicity / teratogenicity - oral/dermal/inhalation: Study was waived; The substance is considered not

to exert any reproductive toxic effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out
C.I. Pigment Violet 23 nanoform

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies:
There are no Studies available for this endpoint
- Available non-GLP studies
There are no Studies available for this endpoint
- Historical human/control data
There are no data available for this endpoint
- (Q)SAR
The test material is outside the applicability domain of these models
- In vitro methods
There are no in vitro methods available for this endpoint
- Weight of evidence
There are no data available for this endpoint
- Grouping and read-across: There is insufficient information available on possible reproductive effects of the registered substance and structural analogues.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
-The study according to OECD TG 414 in the rat is a standard requirement of REACH at the applicable tonnage level. None of the specific conditions set out in column 2 are completely applicable. Nevertheless, we are, based on our knowledge of the test material as well as the whole group of organic pigments, convinced that the test material is insoluble and not bioavailable and that, therefore, the results of this study could be predicted with sufficient confidence. However, we did not succeed in convincing ECHA as this specific situation is not reflected in the standard set by column 2 of the annexes.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
This study will only be performed when deemed necessary after the completion of a study of the same type in the first species

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out
C.I. Pigment Violet 23 nanoform

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies:
There are no Studies available for this endpoint
- Available non-GLP studies
There are no Studies available for this endpoint
- Historical human/control data
There are no data available for this endpoint
- (Q)SAR
The test material is outside the applicability domain of these models
- In vitro methods
There are no in vitro methods available for this endpoint
- Weight of evidence
There are no data available for this endpoint
- Grouping and read-across: There is insufficient information available on possible reproductive effects of the registered substance and structural analogues.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
-The study according to OECD TG 414 in the rat is a standard requirement of REACH at the applicable tonnage level. None of the specific conditions set out in column 2 are completely applicable. Nevertheless, we are, based on our knowledge of the test material as well as the whole group of organic pigments, convinced that the test material is insoluble and not bioavailable and that, therefore, the results of this study could be predicted with sufficient confidence. However, we did not succeed in convincing ECHA as this specific situation is not reflected in the standard set by column 2 of the annexes.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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:

In an oral study according to OECD TG 421 in rats the substance did not exert any reproductive toxic effects

It can reasonably be deduced that Pigment Violet 23 does not cause toxicity to reproduction and thus does not have to be classified according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC), because

- Pigment Violet 23 is a chemically unreactive substance,

- Pigment Violet 23 can be considered insoluble because it has an extremely low solubility in water and n-octanol,

- due to its extremely low solubility, it is unlikely that Pigment Violet 23 becomes systemically bioavailable after oral, dermal or inhalation exposure,

- Pigment Violet 23 caused no systemic toxic effects in a 28-day oral gavage study in rats (NOAEL 1000 mg/kg/day) and there was no evidence of absorption of the substance,

- Pigment Violet 23 does not have to be classified as skin sensitizing or as skin or eye irritating, indicating that its chemical inertness and extremely low solubility in water and n-octanol largely prevent interaction with living cells and tissues.

It can therefore be concluded with sufficient certainty that Pigment Violet 23 will not cause toxicity to reproduction or developmental toxicity a

nd that testing is not scientifically necessary.