[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The objective of this Reproduction/Developmental Toxicity Screening Test in Sprague Dawley Rats with test item Dodicor V 5654 by Oral Gavage in Sprague Dawley Rats as per OECD 421 guidelines, was to evaluate male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition with repeated exposure once daily to males for a period of 30 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 51 to day 63 of treatment).

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 males and 12 females. The animals in group G1 were administered with vehicle [Corn Oil], the animals in G2, G3 and G4 groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low, mid and high doses respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 4 mL/kg body weight.

For reproduction toxicity end points, there were no effects noted in male mating and fertility index of all tested dose groups. No effects were noted in female mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length at all tested dose groups. For maternal toxicity end points, there were no irregularities noted in oestrus cyclicity and no changes in mean cycle length, no changes in mean body weight, percent change in mean body weight gain and mean feed consumption during gestation and lactation periods at all the tested dose groups. There were no changes observed in birth parameters and litter observations during lactation period. The number of implantations, post-implantation and post-natal losses were unaffected by test item at all the tested dose groups.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2020 to 28 July 2020

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:

Adopted on 29 July 2016

Deviations:

no

Principles of method if other than guideline:

Not Applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The objective of this Reproduction/Developmental Toxicity Screening Test in Sprague Dawley Rats with test item Dodicor V 5654 by Oral Gavage in Sprague Dawley Rats as per OECD 421 guidelines, was to evaluate male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition with repeated exposure once daily to males for a period of 30 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 51 to day 63 of treatment).

- Basis for dose level selection:
Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Dodicor V 5654 by Oral Gavage in Sprague Dawley Rats [Bioneeds Study No. BIO-DTX 023] was conducted with the doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control (G1), low dose (G2), mid dose (G3) and high dose (G4), respectively. There were no test item-related effects noted at all the tested dose groups (100, 300 and 1000 mg/kg body weight) for both reproduction and developmental toxicity end points. Based on the results obtained the doses of 0, 100, 300 and 1000 mg/kg body weight were selected as control, low, mid and high dose groups for present Reproduction/Developmental Toxicity Screening Test of Dodicor V 5654 by oral gavage in Sprague Dawley Rats.

- Route of administration:
The vehicle (corn oil) and test item formulations were administered once daily through oral (gavage) route as it is the probable route of human exposure. Hence, oral route was selected for dose administration.

- Other considerations:
Species: Rat,
Strain: Sprague Dawley,
Number of animals: 96 (48 Males + 48 Females)
A total of 58 females were received and evaluated pre-exposure for estrous cyclicity and females that failed to exhibit typical 4 to 5 day cycles were excluded from the experiment and sacrificed on the day of initiation of the treatment by carbon dioxide asphyxiation. Finally, 48 females were selected for the study. Females used were nulliparous and non-pregnant.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Batch produced by : Clariant Ibérica Producción, S.A.
- Lot no.: ESD0033040
- Expiration date of the lot: August 2021
- Purity: UVCB substance; purity ca 97%
- Purity test date: 23 August 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29oC)
- Stability under test conditions: The stability and homogeneity of test item in dose formulations was established by Analytical Department of Bioneeds India Private Limited (Bioneeds study no.: BIO-ANM 1468) before initiation of treatment. The test item formulations were stable and homogenous for 6 hours at room temperature and 48 hours at 2 to 8˚C with concentrations of 5 mg/mL and 250 mg/mL.
However, freshly prepared test item formulations were administered to the animals and homogeneity was achieved by constant and thorough stirring using magnetic stirrer.
- Solubility and stability of the test substance in the vehicle: The test item was not miscible with water or 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) and clearly miscible with corn oil at the concentration of 250 mg/mL (the highest dose concentration selected for the study considering the dose volume of 4 mL/kg body weight) as per in-house miscibility test results.
Hence, corn oil was selected as vehicle for test item formulation preparation. Corn oil is a routinely used vehicle of choice for the oral toxicity studies and universally accepted.

FORM AS APPLIED IN THE TEST: Liquid [Test material was diluted to the required concentration with the vehicle and administered to the animals].

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: (P) 9 wks
- Weight at study initiation: (P) Males: 240.74 to 279.64 g; Females: 210.06 to 250.01 g
- Fasting period before study: No
- 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.
- 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 presence of sperm in the vaginal smear and/or vaginal plugs (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 mated females from gestation day 20 onwards.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimatization period: Males: 20 February 2020 to 09 March 2020;
Females: 20 February 2020 to 24 February 2020 (later females were evaluated for determination of oestrus cyclciity for a period of two weeks)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 to 22.9°C
- Humidity (%): 46 to 66%
- 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: 20 February 2020; To: 10 May 2020

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared daily before dose administration.

The required quantity of test item was weighed into a clean beaker and small volume of the vehicle was added into the beaker, mixed well using glass rod and transferred into measuring cylinder. Rinsing procedure was repeated until complete transfer of test item formulation into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get desired concentration of 25, 75 and 250 mg/mL of test item for low, mid and high dose groups respectively.
The test formulations were maintained under stirring conditions using magnetic stirrer to maintain homogeneity of the test item formulations.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
The test item was not miscible with water or 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) and clearly miscible with corn oil at the concentration of 250 mg/mL (the highest dose concentration selected for the study considering the dose volume of 4 mL/kg body weight) as per in-house miscibility test results. The actual dose volume for each animal was calculated based on the most recent body weight. Hence, corn oil was selected as vehicle for test item formulation preparation. Corn oil is a routinely used vehicle of choice for the oral toxicity studies and universally accepted.
- Concentration in vehicle:
G1 (Vehicle Control): 0 mg/mL; G2 (Low Dose): 25 mg/mL; G3 (Mid Dose): 75 mg/mL; G4 (High Dose): 250 mg/mL
- Amount of vehicle (if gavage): 4 (mL/kg body weight)
- Lot no.: L12017004 and L32011001
-Purity: 99.9 %

Details on mating procedure:

- M/F ratio per cage: 1 : 1
- Length of cohabitation: 13 Days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: Gestation Day 0
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: No unsuccessful mating was found
- Further matings after two unsuccessful attempts: [no / yes (explain)]: No
- After successful mating each pregnant female was caged (how):
After confirming presence of sperm in the vaginal smear and/or vaginal plugs (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 mated females from gestation day 20 onwards.
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and dose formulation analysis was done by Analytical Chemistry department of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 1468 and the results are presented in the report. Sampling and analysis of formulations were performed during week 1and week 5 of the treatment. The samples were collected in duplicates (5 mL each) from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates (5 mL each) 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 aliquot of each formulation was analyzed. The second aliquot was stored as a 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) is ≤10%.

Duration of treatment / exposure:

Once daily to males for a period of 30 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (minimum: 51 days and maximum: 63 days of treatment).

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.:

100 mg/kg bw/day

Remarks:

Low Dose

Dose / conc.:

300 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:

12 males + 12 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose Range Finding Study for Reproduction/ Developmental Toxicity Screening Test of Dodicor V 5654 by Oral Gavage in Sprague Dawley Rats [Bioneeds Study No. BIO-DTX 023] was conducted with the doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control (G1), low dose (G2), mid dose (G3) and high dose (G4) respectively, in consultation with sponsor. There were no test item-related effects noted at all the tested dose groups (100, 300 and 1000 mg/kg body weight) for both reproduction and developmental toxicity end points when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 29 days), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13, under the experimental conditions employed.

Based on the results obtained from the Dose Range Finding Study [Bioneeds Study No. BIO-DTX 023], the doses of 0, 100, 300 and 1000 mg/kg body weight were selected as control, low, mid and high dose groups in consultation with sponsor for present Reproduction/Developmental Toxicity Screening Test of Dodicor V 5654 by oral gavage in Sprague Dawley Rats.

- Rationale for animal assignment (if not random): The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ±20% (Males: -14.92 to 10.88%; Females: -14.67 to 8.42%) of the mean body weight of each sex. The grouping was done one day prior to the initiation of treatment by randomization based on the body weights. Body weight of the animals were analyzed statistically for mean body weight to rule out statistical significant differences between groups within each sex.

Positive control:

Not Applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily for clinical signs and twice daily for mortality.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: The animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period and on day 14 (fasting body weight).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

Oestrus cyclicity was monitored for two weeks after the five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the 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 (lactation day 14).

Sperm parameters (parental animals):

Parameters examined in [all/P] male parental generations:
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, 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 determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All animals were sacrificed after completion of 30 days of treatment (on Day 31)
- Maternal animals: All animals were sacrificed on lactation day 14 and pups were sacrificed on PND 13. The females confirmed with mating but not littered were sacrificed on gestation day ‘25’.

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 [15] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The dead pups and pups which were sacrificed on PND 4/13 were examined for gross abnormalities with particular attention to the external reproductive genitals and 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 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 the 95% level of confidence (P<0.05).
Note: Data of non-pregnant females were presented in the individual animal data but excluded for mean calculations and statistical analysis. The data of females mated but not littered and lactation data of females with total litter loss were presented in individual animal data and considered for mean calculations and statistical analysis where applicable.

Parametric - One-way ANOVA with Dunnett’s post test was followed for the following parameters:
• Body weight (weekly body weights and gestation/lactation body weights)
• Percent Change in body weight (weekly body weights and gestation/lactation body weights)
• Feed consumption (weekly and 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 (adults/pups)

Non parametric - Kruskal-Wallis was followed for the following parameters
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter at birth and during lactation period
• Litter size at birth and during lactation period
• No. of early resorptions/litter
• No. of late resorptions/litter
• Pups abnormalities (if any) per litter
• Post-implantation loss/litter
• Postnatal loss/litter

Cross Tabs - Chi-square test was followed for the following parameters
• Pregnancy rate
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions

Reproductive indices:

Male Mating Index (%):
No. of males with evidence of mating / Total no. of males used for mating X 100
Male Fertility Index (%):
No. of males siring a litter / Total no. of males used for mating X 100
Female Mating Index (%):
No. of females with evidence of mating (or confirmed pregnancy) / Total no. of females used for mating X 100
Female Fertility Index (%):
No. of females with confirmed pregnancy / Total no. of females used for mating X 100
Pre-coital Interval (Days):
Date of confirmation of mating – Date of initiation of cohabitation
Mean Gestational Length:
Date of parturition – Date of positive evidence of mating (GD 0)
Gestation Index (%):
Number of females with live born / Number of females with evidence of pregnancy X 100
Parturition Index (%):
No. of females littered / No. of females with evidence of pregnancy X 100
Mean number of Implantations (%):
Sum of number of implantations in the group / Total no. of dams in the group X 100
Post-Implantation Loss (%)/Litter:
No. of Implantations - No. of Viable pups / No. of Implantations X 100
Post-implantation Loss/litter (No.):
No. of implantations- No. of live births
Post-natal Loss on lactation day 13 (No.):
Live births - pups alive at lactation day 13
Post natal loss on lactation day 13 (%):
No. of pups alive on postnatal day 13 / No. of live pups at birth X 100

Offspring viability indices:

Live Birth Index (%) per dam:
Number of pups born alive / Total number of pups born X 100
Pup Survival index (%) on lactation day 4/7/13:
Total number of live pups (on lactation day 4/7/13) / Number of pups born/on LD 4/on LD 7 X 100
Sex ratio (M/F):
Number of male offspring / Number of female offspring
Percentage of male/female offspring (%):
Number of male/female offspring / Total number of offspring X 100
Ano-genital Distance to cube root of the body weight Ratio (AGD ratio):
Ano-genital Distance (mm) / Cube root of the body weight X 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity noted in either sex at all the tested dose groups [G2 (100 mg/kg body weight/day), G3 (300 mg/kg body weight/day), G4 (1000 mg/kg body weight/day)] and vehicle control group [G1 (0 mg/kg body weight/day] throughout the experimental period.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity noted in either sex at all the tested dose groups [G2 (100 mg/kg body weight/day), G3 (300 mg/kg body weight/day), G4 (1000 mg/kg body weight/day)] and vehicle control group [G1 (0 mg/kg body weight/day] 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 with respect to day 1 in either sex during the experimental period at any of the tested dose groups when compared with vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean feed consumption measured during pre-mating period (first 2 weeks) in either sex at all the tested dose groups when compared with vehicle control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related histopathological findings in high dose group animals of both sexes.
A single incidence of focal epithelial vacuolation in epididymides and mild degeneration of seminiferous tubules in testes observed across the groups were considered to be incidental findings.

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) hormone levels: There were no changes observed in serum Thyroxine (T4) hormone levels at all the tested dose group males when compared with vehicle control group.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no irregularities observed in the oestrus cyclicity of females at any of the 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 at all the tested dose groups when compared with vehicle control group.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male Mating Index (%):
A total of 12 (out of 12) males were confirmed with a mating index of 100.0% from all the tested dose groups G2, G3, G4 and vehicle control group G1 respectively. There were no statistically significant differences noted in male mating index (no. of males impregnating females) at any of the tested dose groups when compared with vehicle control group.

Male Fertility Index (%):
A total of 11 (out of 12), 10 (out of 12), 11 (out of 12) and 11 (out of 12) males were confirmed with impregnating a female with a fertiltiy index of 91.7%, 83.3%, 91.7% and 91.7% from group G1, G2, G3 and G4, respectively. There were no statistically significant differences noted in male fertiltiy index (no. of males impregnating females) at any of the tested dose groups when compared with vehicle control group.

Female Mating Index (%):
All the 12 females from each tested dose group (G2, G3 and G4) and vehicle control group (G1) were confirmed with mating with a mating index of 100% for each group. There were no statistically significant differences noted in female mating index (no. of females with evidence of mating) at any of the tested dose groups when compared with the vehicle control 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 group G1, G2, G3 and G4, respectively. There were no statistically significant effects noted in female fertility index at all 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 is 4.58, 6.25, 3.58 and 6.67 days for groups G1, G2, G3 and G4, respectively. There was no statistically significant difference 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] is 22.09, 22.10, 22.00 and 22.27 days for groups G1, G2, G3 and G4, respectively. There was no statistically significant difference 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%, 100%, 100% and 100% from group 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 live born pups with a pregnancy index of 91.7%, 83.3%, 91.7% and 91.7% from group G1, G2, G3 and G4, respectively. There was no statistically significant difference 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 is 11.00, 11.90, 10.64 and 12.00 from group G1, G2, G3 and G4, respectively. There was no statistically significant difference for this parameter in any of the tested dose groups when compared with the vehicle control group.

Post implantation loss per litter (No.) and (%):
The post implantation losses per litter is 0.36, 0.50, 0.64 and 0.36 with a percentage of 2.76, 4.00, 7.20 and 2.59 respectively from group G1, G2, G3 and G4, respectively. There was no statistically significant difference for this parameter in any of the tested dose groups when compared with the vehicle control group.

Post-natal losses on Lactation Day 13 (No.) and (%):
There was no post-natal loss noted in any of the litters from all the tested dose and vehicle control groups.

Percentage of male/female offspring per litter (%):
The percentage of male and female offspring per litter is 49.75 and 50.25 for G1, 52.48 and 47.52 for G2, 41.74 and 58.26 for G3 and 58.94 and 41.06 for G4 respectively. There was no statistically significant difference for this parameter in any of the tested dose groups when compared with the vehicle control group.

Note: The data of 11 (out of 12 mated females), 10 (out of 12 mated females), 11 (out of 12 mated females) and 11 (out of 12 mated females) females confirmed with parturition from group G1, G2, G3 and G4, respectively was considered for mean calculations and subjected to statistical analysis to analyse the maternal end points.

Key result

Dose descriptor:

NOAEL

Effect level:

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 consumption and compound intake

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 at all the tested dose groups and vehicle control group litters during post-natal 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 were noted during daily observation of pups at all the tested dose groups and vehicle control group litters during post-natal period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes observed in mean pup [both male and female] weight per litter recorded on post-natal day (PND) 1, 4, 7 and 13 at all the tested dose group litters when compared with vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during necropsy in any of the pups of both sexes at all the tested dose groups and vehicle control group.

Other effects:

no effects observed

Description (incidence and severity):

Serum Thyroxine (T4) hormone levels: There were no changes observed in serum Thyroxine (T4) hormone levels of PND 13 pups from all the tested dose group litters when compared with vehicle control group.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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 Thyroxine (T4) Hormone Levels

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, the oral administration of the test item Dodicor V 5654 over a relatively limited period of time (for males with a total of 30 days and for females with a maximum period of 63 days) did not produce any indication of reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of test item Dodicor V 5654 is considered to be 1000 mg/kg body weight for reproduction and developmental toxicity end points.

Executive summary:

The objective of this Reproduction/Developmental Toxicity Screening Test in Sprague Dawley Rats with test item Dodicor V5654 by oral gavage as per OECD 421 guideline was to evaluate male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition with repeated exposure once daily to males for a period of 30 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 51 to day 63 of treatment).

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 males and 12 females. The animals in group G1 were administered with vehicle [Corn Oil], the animals in G2, G3 and G4 groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low, mid and high doses respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 4 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item was stable up to 6 hours at room temperature and 48 hours at 2 to 8^˚C in corn oil at the concentration of 5 mg/mL and 250 mg/mL.Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1 and 5 of the dose administration period 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 animals were observed once daily for clinical signs, twice daily for mortality and morbidity. The weekly body weight and weekly feed consumption was recorded once weekly (except during cohabitation) for all the animals. The serum thyroxine hormone (T4) levels were estimated for all males by ELISA method. The gross pathology and organ weighing were performed on the day of termination for all animals. Detailed histopathological examination was conducted on ovaries, testes and epididymis from the groups G1 and G4 animals with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure.

All the males were evaluated for reproductive performance such as, mating and fertility index. All the litters were evaluated for reproductive performance such as, mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length. All the females were evaluated for oestrus cyclicity during, premating, mating treatment period and at termination. The body weight and feed consumption was recorded for all the females during gestation days 0, 7, 14 and 20 and on lactation days 1, 4, 7 and 13. Terminal body weight was measured on the day of the termination (LD 14). At birth / litter observation parameters such as, number of live/dead pups born, litter size, sex ratio, live birth index per litter and pup survival index were observed / calculated for all the litters. The total number of implantations per litter was recorded during necropsy and the post-implantation and post-natal losses were calculated.

The pups were observed once daily for external examinations and twice daily for mortalities till termination [post-natal day (PND) 13], weighed individually on PND 1, 4, 7 and 13, measured for ano-genital distance (AGD) to calculate AGD ratio on PND 4, observed for retention of any nipples/areolae in male pups on PND 13. All the pups were observed for gross pathological observations at termination and analyzed the serum collected from PND 13 pups for thyroxine hormone (T4) levels by using ELISA method.

There were no clinical signs of toxicity and no mortality/morbidity noted at all the tested dose group animals. There were no changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption at all the tested dose groups of both the sex. There were no changes noted in mean serum thyroxine hormone (T4) levels at all the tested dose group males. The absolute and relative organ weights did not reveal any changes at all the tested dose groups. No macroscopic changes noted during conduct of necropsy at all the vehicle control and tested dose groups of both the sex. Histopathological examination conducted for group G4 animals of both sexes did not reveal any test item related microscopic changes.

For reproduction toxicity end points, there were no effects noted in male mating and fertility index of all tested dose groups. No effects were noted in female mating index, fertility index, gestation index, parturition index, pre-coital interval and gestation length at all tested dose groups. For maternal toxicity end points, there were no irregularities noted in oestrus cyclicity and no changes in mean cycle length, no changes in mean body weight, percent change in mean body weight gain and mean feed consumption during gestation and lactation periods at all the tested dose groups. There were no changes observed in birth parameters and litter observations during lactation period. The number of implantations, post-implantation and post-natal losses were unaffected by test item at all the tested dose groups.

For developmental toxicity end points, there were no external anomalies noted and no test item-related mortalities noted in pups during post-natal period at all the tested dose group litters. There were no test item-related changes noted in mean pup weight and mean pup ano-genital distance ratio per litter in either sex at all the tested dose groups. There were no occurrences of nipples in male pups examined on PND 13 from all the tested dose and vehicle control group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested and control group litters. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 (from all litters) pups at all the tested dose group litters.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restriction, OECD 421 Guideline conform study.

Effects on developmental toxicity

Description of key information

An OECD 421 Reproduction/Developmental Toxicity Screening Test in Sprague Dawley Rats with test item Dodicor V 5654 by Oral Gavage in Sprague Dawley Rats study is available.

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 males and 12 females. The animals in group G1 were administered with vehicle [Corn Oil], the animals in G2, G3 and G4 groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low, mid and high doses respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 4 mL/kg body weight.

Males were treated two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period). Females were treated for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 (day 51 to day 63 of treatment).

For developmental toxicity end points, there were no external anomalies noted and no test item-related mortalities noted in pups during post-natal period at all the tested dose group litters. There were no test item-related changes noted in mean pup weight and mean pup ano-genital distance ratio per litter in either sex at all the tested dose groups. There were no occurrences of nipples in male pups examined on PND 13 from all the tested dose and vehicle control group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested and control group litters. There were no changes noted in serum thyroxine (T4) hormone levels estimated for PND 13 (from all litters) pups at all the tested dose group litters.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restriction. OECD 421 guideline conform study.

Justification for classification or non-classification

The oral administration of the test item Dodicor V 5654 over a relatively limited period of time (for males with a total of 30 days and for females with a maximum period of 63 days) did not produce any indication of reproduction and developmental toxicity at the dose levels of 100, 300 and 1000 mg/kg body weight/day under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of test item Dodicor V 5654 is considered to be 1000 mg/kg body weight for reproduction and developmental toxicity end points.

According to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006 the test substance Dodicor V 5654 has not to be classified for reproduction/developmental toxicity.