N,N-bis(2-hydroxypropyl)benzamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Daily oral (gavage) administration of Primid V 40-32 to male and female Wistar rats at dose levels of 200, 500 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters. The No Observed Adverse Effect Level (NOAEL) of Primid V 40-32 for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan-Jul 2017

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:

2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The purpose of this study in Wistar Rats was to generate limited information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

Specific details on test material used for the study:

Test item commercial name: Primid V 40-32
Physical appearance: White powder
Batch No.: 9115151/08

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HanTac: WH

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

Age at treatment: 13-14 weeks

Body weight range at the start of treatment:
Males: 327.44 to 369.29g
Females: 211.43 to 247.49g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.

Identification : Temporary: All animals were identified using cage card and crystal violet solution body marking during acclimatization and pre-treatment periods.Permanent: All rats were identified using rat accession number indicated on the cage card and tail tattooing with the numerical details of the rat accession number only. Tail tattooing was done two days after grouping. Turmeric body marking was done on the day of grouping during pre-treatment period for permanent identification and animals were not re-marked thereafter.
Pups were identified using nail clipping method.

Acclimatization: After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days. During the acclimatization period, animals were observed once daily for any abnormalities. Only the animals that are determined by the veterinarian were assigned to this study. Female rats used in this study were nulliparous and non-pregnant.


Husbandry

Environmental Conditions

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 20−25°C and relative humidity between 50 - 68%. The photoperiod was 12 hours’ light and 12 hours’ dark cycle. Adequate fresh air supply of 12 - 15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Housing
Pre-mating:
Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes.
Mating and post-mating:
During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.
Enrichment: Polycarbonate rat huts were provided to the animals as environmental enrichment objects during pre-mating period and post-mating period for males and pre-mating period for females. Enrichment was changed along with cage at least once a week.

Bedding
Steam sterilized clean corn cob was used as bedding and changed along with the cage at least twice a week.
Contaminant analysis report for bedding material is presented in Annexure 1 of the full study report.

Diet and water
Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet (Certified) manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, WI 53744-4220, was provided ad libitum to animals. A sample of the diet was retained and was discarded.
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filter-cum-purifier manufactured by Eureka Forbes Limited., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
The food and water provided to the animals were tested for contaminants. Analysis and contaminant analysis reports of food and water are included as Annexures 2 to 4 of the full study report.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

Males: The dose formulation was administered orally by gavage to specific group of rats at approximately the same time each day (varying by ± 3 hours), for a period of 34 days (which includes two weeks prior to mating, during the mating period and post mating) after which they were sacrificed after overnight fasting.
Females: The dose formulation was administered orally by gavage to the specific group of rats at approximately the same time each day (varying by ± 3 hours), two weeks prior to the mating period and was continued through mating, pregnancy and up to LD 13. On LD 14, the females were sacrificed after overnight fasting.
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only.
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control group at an equivolume of 10 mL/kg Bwt.

Details on mating procedure:

Females were placed with a single male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and /or vaginal plug. Subsequently, pregnant females were housed individually until LD 14.
All the mated females were maintained till they litter or for a maximum period of at least 25 days from the last day of cohabitation. Not littered females were sacrificed after 25 days from the day they are found of sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration, stability and homogeneity of the test item in the vehicle were established at 0.9 and 110 mg/mL under Advinus Study No. G12546. The dose formulation concentrations were found to be within the acceptance limit of ±15% of target and % RSD for homogeneity was less than 10%.Based on the results, the test item was stable and homogenous in the vehicle up to 24 hours when stored at room temperature.

Duration of treatment / exposure:

Males: 34 days (which includes two weeks prior to mating, during the mating period and post mating)
Females: two weeks prior to the mating period and was continued through mating, pregnancy and up to LD 13. On LD 14.
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only.

Frequency of treatment:

Males: approximately the same time ach day (varying by ± 3 hours)
Females: approximately the same time each day (varying by ± 3 hours)
The animals in the vehicle control group were handled in an identical manner to the treatment group and were administered vehicle only.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle Control group (G1)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Low dose group (G2)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Mid dose group (G3)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose group (G4)

No. of animals per sex per dose:

No. of groups: 4
G1: Vehicle Control
G2: Low Dose
G3: Mid Dose
G4: High Dose
Main groups: 10 males + 10 females per group
Total = 80 (40 males + 40 females)

Control animals:

yes, concurrent vehicle

Positive control:

no

Parental animals: Observations and examinations:

Clinical Signs, Morbidity and Mortality
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs, the observation for morbidity and mortality was carried out once in the morning during holidays. All rats were observed for clinical signs once daily during the treatment period.

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

Body Weights
i)Individual body weights of males were recorded initially and at weekly intervals up to week 4 and on day 34 of the treatment period. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii)All dams were weighed on GD 0, 7, 14 and 20 and on LD 0, 4 and 13.

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

Oestrous cyclicity (parental animals):

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

Sperm parameters (parental animals):

Not examined

Litter observations:

Litters
a. Each day morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded.
c. On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Blood samples were collected from two of the surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels. Partial adjustment was done whenever the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size dropped below the culling target (8 pups/litter). From the available surplus pups (above the culling target), blood was collected for serum T4 assessments.
d. After standardization, the individual pup body weight was recorded on Day 13 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from cube root of body weight.
f. The number of nipples/areolae in male pups was counted on PND 13.
g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
h. The litters were observed daily to note the number of alive, dead and cannibalized pups.
i. In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded.
j. Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated

Postmortem examinations (parental animals):

Gross Necropsy
All adult animals were examined macroscopically for any structural abnormalities or pathological changes. The adult animals killed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The rats were subjected to detailed necropsy.
For apparently non-pregnant rats, the uteri were stained with Salewski stain to identify the pre-implantation loss of the embryos.
The number of implantation sites was recorded for all the dams.

Tissue Collection
On completion of the gross pathology examination the tissues and organs noted below were collected and weighed from all adult animals. The organ weight ratios (organ to body weight) as percentage of fasting body weight was determined and presented in the report. The paired organs were weighed together and combined weight was presented. The tissues were preserved in 10% Neutral Buffered Formalin (NBF) unless noted otherwise.

Tissue/Organs Organ Weighing Collection and Preservation Microscopic Examination
All gross lesions x x
Epididymides x x x
Ovaries with oviducts x x x
Thyroid with parathyroids@ x x x
Uterus with cervix x x x
Vagina x x
Prostate x x
Seminal vesicles and
coagulating glands x x
Testes* x x x
Levator ani bulbocavernosus
muscle complex x x
Cowper’s glands x x
Glans penis x x

* : Collected in modified Davidson’s fluid
X: Activity performed.
@: Weighed after formalin fixation


Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. In addition, the testes were also subjected for qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structures. All gross lesions were examined in all the groups. In the absence of any test item-related findings at the high dose, the low and mid dose groups were not evaluated. The reproductive organs from not littered rat (Rt8126; low dose group) were also examined.
The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues will be archived.

Postmortem examinations (offspring):

Gross Necropsy
All pups were examined macroscopically for any structural abnormalities or pathological changes. All survived pups were necropsied on lactation Day 13. Particular attention was paid to the external genitals which were examined for signs of altered development. Dead and moribund pups were also examined for possible defects and/or cause of death.

Tissue Collection
On LD 13, thyroid gland from available one male and female pup from each litter (randomly selected) was collected and preserved in 10% NBF for the histopathological examination. The thyroid weight was determined after fixation

Histopathology
The available thyroid gland from a male and a female pup per litter (randomly selected) were evaluated from all the groups.
The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin Eosin stain. In addition, testes were sectioned at 3-4 µm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues will be archived.

Statistics:

Statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Version 12.0. All data of quantitative variables like body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance, ano-genital index and organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-Way Analysis of Variance (ANOVA). When the data found to be non-optimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test when the overall treatment ‘F’ test is found significant.
Post implantation loss (%), no. of implantations, pre-coital interval (days), mean litter size, sex ratio and gestation length (days) were analysed after suitable transformation (√ x + 1/2) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was performed for testing the differences found significant.
Z test was performed for testing the differences in proportions for mating, fertility and survival indices.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the tests were designated throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the vehicle control group

Reproductive indices:

Reproductive Performance Data of Parents
a.Male mating index (%)
Number of males with evidence of mating=x 100 Number of males cohabited

b.Male fertility index (%)
=Number of males siring a litter/Number of males cohabited x 100


c.Female mating index (%)
=Number of females mated/Number of females cohabited x 100

d.Female fertility index (%)
Number of pregnant females (confirmed at necropsy)
=x 100 Number of females used for mating

e.Mean number of implantations/group
=Total number of implantations /Total number of pregnant animals

f.Post implantation loss (%)
= Number of implantations - Number of live pups/Number of implantations x 100

Offspring viability indices:

a.Mean litter size per group
=Total Number of pups/ Total Number of littered animals

b.Mean viable litter size
=No. of viable pups on Day 1/ No. of females littered

c.Live birth index (%)
=No. of viable pups born (at first observation)/Total no. of pups born (at first observation) x 100

d.Day 4 survival index (%)
=Number of viable pups on lactation Day 4/Number of viable pups born x 100

e.Sex Ratio (%)
=No. of male pups born/Total No. of pups born x 100


f.Ano-genital Distance Ratio (mm/g1/3 )
=Ano-genital distance / Cube root of body weight

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs observed during the treatment period at all the treated groups in both sexes.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

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

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not altered throughout the treatment period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control. However, incidence of significantly higher food consumption was observed during Days 29-34 in males at 1000 mg/kg Bwt/day dose. This was considered toxicologically not significant as there were no significant differences observed in mean body weights

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No eye effections were observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no clinical signs observed during the treatment period at all the treated groups in both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological changes in the reproductive organs of parental male and female rats.
There were no test item-related microscopic changes observed in thyroid gland of parents and pups of all the dose levels.
The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.
All other microscopic findings observed across the groups in male and female rats were considered as incidental changes.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Single incidence of unilaterally enlarged kidneys in 500 mg/kg dose adult females was histologically associated with nephroblastoma and was considered incidental.

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation).
The calculated mean oestrous cycle length was 3.94, 4.06, 4.03 and 4.06 days in vehicle control, 200, 500 and 1000 mg/kg Bwt/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The duration of gestation (gestation length), pre-coital time, fertility indices were not affected at any of the doses tested, when compared to vehicle control group. Incidence of significantly lower male and female fertility index (90%) at 200 mg/kg Bwt/day was observed. This significant difference was considered to be incidental as the difference was not dose-related.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses. However, incidence of significantly lower Day 4 survival index was observed at 500 (82.2%) and 1000 (83.1%) mg/kg Bwt/day doses. This significant difference was within the historical control data (HD range: 79.1% to 100%). Hence, considered as toxicologically insignificant.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A significant effect on TSH levels was noted in pups. The alteration in TSH activity noted in pups (LD4 and LD13) was attributed to administration of test item. However, the T4 levels remained unaffected. Further, the hormone profile was not affected in parental males and females when compared to the concurrent control group.
In absence of concurrent derangement in T4 levels and the microscopic findings in thyroids, the changes in TSH levels observed in male and female pups were considered to be non-adverse effects of the test item.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no significant changes in terminal body weights and thyroid weights in male/female pups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross pathological examination of dead pups and live pups sacrificed on LD 13 did not reveal any abnormalities at all the dose levels tested.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic changes observed in thyroid gland of pups of all the dose levels

Other effects:

no effects observed

Description (incidence and severity):

No changes attributable to test item were detected in the AGD, body weight and ratio of AGD to the cube root of body weight of either sex. However, an incidence of significantly higher mean weight of male pups at 500 mg/kg Bwt/day dose and female pups at 500 and 1000 mg/kg Bwt/day doses on the day of ano-genital distance measured and significantly lower ano-genital distance in female pups at 1000 mg/kg Bwt/day were observed. The significant difference in mean weight of pups observed was considered incidental as the mean weight of pups per litter were compared to control. The lower ano-genital distance observed at 1000 mg/kg Bwt/day was considered incidental as there was no dose correlation and the decrease was marginal. Significantly lower ratio of AGD to the cube root of body weight was observed in female pups at 500 and 1000 mg/kg Bwt/day. The decreased AGD could be due to increased pup weight at the 500 and 1000 mg/kg Bwt/day and hence, considered as toxicologically insignificant.
The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Key result

Reproductive effects observed:

no

Conclusions:

Daily oral (gavage) administration of Primid V 40-32 to male and female Wistar rats at dose levels of 200, 500 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters. The No Observed Adverse Effect Level (NOAEL) of Primid V 40-32 for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.

Executive summary:

The purpose of this study in Wistar Rats was to generate limited information concerning the effects of Primid V 40-32 on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was weighed and dissolved in vehiclei.e.,0.5% Carboxy methylcellulose Sodium salt (medium viscosity) in Milli-Q Water and administered by oral gavage at the dose levels of 200 (G2), 500 (G3) and 1000 (G4) mg/kg Bwt/day to male and female Wistar rats, at the dose volume of 10 mL/kg Bwt/day. Similarly, vehicle was administered to rats in the vehicle control group. Each group consists of 10 males and 10 female rats. The prepared dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods for males, prior to mating, during conception and pregnancy and after delivery up to Lactation Day (LD) 13 for females

The identity of Primid V 40-32 was provided by the study Sponsor by a Test report (LIMS-No.:144716). The stability and homogeneity of the test item in the vehicle was established at 0.9 and 110 mg/mL under Advinus Study No. G12546. Based on the results, the test item was stable and homogenous in the vehicle (0.5 % Carboxy methylcellulose sodium salt (medium viscosity) in Milli-Q^®water) up to 24 hours when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during 2^ndmonth of the treatment period. The results indicated that the analysed concentrations were within ± 15% of variations from the theoretical concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period where food consumption was not measured. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4, 7 and 13. The number, weight, survival and mortality of pups were recorded during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were examined for the appearance of nipples/areolae on post-natal day (PND 13). Blood samples were collected for thyroid hormone analysis prior to necropsy from males and females (LD 13), on LDs 4 and 13 from available pups. The animals were subjected to detailed necropsy at sacrifice and study plan specified tissues were collected. A gross necropsy was performed on all dams on LD 14 and study plan specified tissues were collected. All the surviving pups were sacrificed on LD 13 and thyroid gland

from available one male and one female pup from each litter was collected for histopathological examination.

Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups. In the absence of any test item-related findings at the high dose, the low and mid dose groups were not evaluated. The reproductive organs from not littered rat (Rt8126; low dose group) were also examined. The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.

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

·        There were no treatment-related clinical signs observed at any of the doses tested in both sexes.

·        The body weights and food consumption were unaffected by the treatment at all the tested doses in both sexes.

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

·        Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, fertility indices of sires and dams and survival indices at all the tested doses.

·        Mean number and weight of male, female and combined sex pups in treated groups were comparable to control group.

·        There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups.

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

·        No areola/nipple retention in male pups on PND 13 at any of the doses tested.

·        There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in males and females at all the tested doses.

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

·               Treatment-related decrease in TSH level in pups (LD4 and LD13) was observed. In the absence of any associated findings, the hormonal alteration was considered as non-adverse effect of the test item.

·        There were no test item-related gross and microscopic changes observed in the reproductive organs and thyroid gland of male and female parents and pups at all the tested doses.

Daily oral (gavage) administration of Primid V 40-32 to male and female Wistar rats at dose levels of 200, 500 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters. The No Observed Adverse Effect Level (NOAEL) of Primid V 40-32 for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.

·        There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in males and females at all the tested

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

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

Daily oral (gavage) administration of Primid V 40-32 to male and female Wistar rats at dose levels of 200, 500 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and post mating in males or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery in females did not induce any adverse effects on fertility, reproductive performance and on the offspring parameters. No developmental toxicity was observed. The No Observed Adverse Effect Level (NOAEL) of Primid V 40-32 for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

The substance did not show any potential to induce any adverse effects on fertility, reproductive performance and on the offspring parameters on the basis of results from one guideline studies, considered valid.

The No Observed Adverse Effect Level (NOAEL) of Primid V 40-32 for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day

Therefore, the substance did not meet the criteria for classification and labelling under reproductive toxicity set out in Regulation (EC) No 1272/2008.