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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March-Oct 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: >98%
- Test substance receipt: with the order dating from 1986-05-13
- Stability under test conditions: Oxydation from air-bound oxygen in aquatic solution;
Specific details on test material used for the study:
Batch number: 20161213
Purity:99.3%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Rats were maintained in an environmentally controlled room. The photoperiod was 12 hours light and 12 hours darkness, fluorescence light hours being 06.00 - 18.00 hours.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Dose levels have been selected based on the results of the dose range finding (DRF) study (JRF Study N° 615-1-04-21396). The following dose levels were selected for the present study in consultation with the Sponsor: 5, 25 and 125 mg/kg b. wt./day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
by analytical method, validated in JRF Study N° 228-2-13-21395. The test item is stable for 8 hours. The active ingredient (a.i.) concentration and homogeneity of the test item were performed before the start of treatment and twice during the treatment period
Duration of treatment / exposure:
42 days
Male: two weeks prior to mating, during the mating (two weeks) and until approximately 80% of the females have delivered. Total days of treatment was 42 days.
Female: two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery.
Recovery male and female: Treatment was given till the first scheduled sacrifice of lactating female rats and then observed further for a period of 14 days without any treatment.

Frequency of treatment:
Dose formulations and the vehicle were administered to male and female rats daily up to and including the day before scheduled sacrifice.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 for treated (15 for each concentration:0-5-25-125 mg/kg bw/day)
5 for control (0-125mg/kg bw/day)
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Mortality and Morbidity: Rats were observed daily, twice, for mortality and morbidity.

Clinical Sign: Rats were observed daily twice, during treatment and recovery periods, for any visible clinical signs.

Bodyweight: was recorded on the first day of dosing and at weekly intervals thereafter.

Food Consumption: Food weights of male rats were determined weekly during the pre-mating and post-mating periods.
In female rats, during the pre-mating period, food weights were recorded at weekly intervals. During the gestation period, food weights were measured on days 0, 7, 14, and 20. During the lactation period, food weights were measured on days 0, 4, 7, and 14. Additional food was offered as and when required.
Food consumption was not measured during the mating period.
Food weights of male and female rats belonging to recovery groups were determined weekly throughout treatment and recovery periods.

Estrous Cycle: Estrous cycle length and pattern were evaluated by vaginal smears of individual female rats during the pre-treatment period of two weeks. Vaginal smear was monitored daily from the beginning of the treatment period until evidence of mating. Vaginal smear, from each pregnant animal, was also observed on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.

Neurobehavioural Observation (NBO):To assess the behavioural and neurological status of each rat, the below-mentioned parameters of NBO were evaluated prior to initiation of treatment and at weekly intervals, thereafter.

Home Cage Observation:
Parameters Observations
Posture Curled up often asleep (Asleep)
Vertical jumping
Writhing (twisting, squirming or contorted motion)
Flattened, limbs may be spread out
Rearing
Sitting normally, feet tucked in (Sitting B)
Sitting but with head hung down (Sitting A)
Lying on side
Circling
Sitting or standing alert, watching (Sitting C)
Clonic Movement Present
Absent
Tonic Movement Present
Absent



Handling Observation
Parameters Observations
Ease of removing animal from cage Very easy (sits quietly) – V.easy
Easy (vocalisations without resistance)
Moderately difficult (rears)
Difficult (runs around cage)
Very difficult (aggressive, attempts to bite with or without vocalisations)
Handling reactivity Difficult (squires, twists, attempts to bite with or without vocalisations)
Freezes (rigid in hand and totally inactive)
Moderately easy (vocalisations without resistance)
Easy (alert, limbs put against the body)
Palpebral closure Eyelids slightly closed
Ptosis - drooping of eyelids half
Eyelids completely closed
Eyelids wide open (W.open)
Lacrimation None (no external lacrimation)
Severe (drooping of tears)
Slight (wetness of lower eyelids)
Moderate (wetness of eyelids and its surrounding area)
Eye examination Normal
Discharge (draining of normal or pathological content)
Conjunctivitis (inflammation of conjunctival mucus membrane)
Chemosis (swelling of the conjunctiva)
Cataract (opacity of lens)
Corneal opacity (an opaque spot or area on cornea)
Microphthalmos (abnormally small eyeball)
Exophthalmos (abnormal protrusion of the eyeball)
Piloerection Absent
Present
Skin examination Alopecia (absence or lose of hair)
Rough coat (ungroomed or greasy hair coat)
Dermatitis (inflammation of skin)
Normal
Salivation Severe (drooping of saliva)
Moderate (wetness of lower mandible and its surrounding areas)
Slight (wetness of lower mandible)
None (no external salivation)

Open Field Observation: For open-field observations, rats were placed (one at a time) in an open arena (size: 495× 495 × 280 mm) with a flat surface covered with clean absorbent paper and observed for a period of 3 minutes. During the 2 minutes period, each rat was observed for the below-mentioned parameters:

Parameters Observations
Gait Normal
Slightly abnormal
Moderately abnormal
Severely abnormal
Mobility Slightly impaired
Moderately impaired
Normal
Totally impaired
Arousal Very low (V.low)
High
Low
Very high (V.high)
Vocalisations Vocalisations (actual number)
Rears Rears (actual number)
Respiration Snuffles (accumulation of secretion with respiratory noise)
Normal
Dyspnoea (abnormally difficult or laboured breathing)
Tachypnoea (quick and usually shallow respiration)
Abdominal breathing (breathing by diaphragm)
Gasping (convulsive catching of breath with wide open mouth)
Clonic movement Mild clonic tremors of limbs
Absent
Chewing, clonus of the jaws
Repetitive clonic tremors of the whole body
Tonic movement Opisthotonos (head, body and limbs rigidly arched backward)
Absent
Tonic contraction or extension of hind limbs
Emprosthotonos (head, body and limbs extended forward)
Urination Urination (actual number of urine pool in open field)
Defecation Defecation (actual number of fecal bolus in open field)
Stereotypy Absent
Circling
Excessive grooming
Other (actual observations)
Bizarre behavior Other (actual observations)
Self-destructive biting (e.g., tail, paws) or self-mutilation
Biting of open field/standard arena
Absent
Retropulsion (moving backward)




Functional Observational Battery (FOB) : The below mentioned FOB parameters along with NBO parameters were performed from randomly selected five rats/sex/group towards the end of the study.



Sensory Reactivity Measurements: For sensory reactivity measurements, rats were placed in an open arena (size: 495 × 495 × 203 mm) with a flat surface covered with clean absorbent paper. The below-mentioned parameters were performed and recorded for rats.
A) Approach Response
Each rat was approached at nose level with the end of a blunt object. The object was held approximately 3 cm away from the face of the rat for approximately 4 seconds to allow time for the rat to respond. The degree of the elicited response was recorded as absent, slow, moderate or fast response.
B) Touch Response
Approaching the rat from the side, the rump of the rat was gently touched with a blunt object. The contact was brief (approximately 1 to 2 seconds) and deliberate but not forceful. The degree of the elicited response was recorded as the absent, slight, normal or exaggerated response.
C) Click Response
A clicker was positioned approximately 5 cm above the back of the rat with care taken not to have the clicker in the rat’s field of vision. The clicker was held in the palm of the hand to ensure consistency of sound from test to test. The degree of the elicited response of the rat to the click sound was recorded as the absent, slight, normal or exaggerated response.
D) Pupil Response
The beam of a pocket-sized flashlight was brought from a lateral position medially towards the centre of the face of the rat. Constriction of the pupil was observed as a positive response. The degree of elicited response was recorded as normal or abnormal.
E) Tail-pinch Response
The tail was squeezed approximately 2 to 3 cm from the tip using forceps (always applying about the same amount of force for each rat). The degree of the elicited response was recorded as absent, slight, flinch (normal) or exaggerated response.
F) Air Righting Reflex
Each rat was held supine, with the hands of the observer under the back and shoulders of rat for support. The rat was dropped from a height of approximately 30 cm. The ease and uprightness of the landing were recorded as normal, slightly abnormal, moderately abnormal or severely abnormal.

Hind Limb Foot Splay: The landing hind limb feet of each rat were marked with a non-permanent, non-toxic ink just prior to testing. Each rat was suspended in a prone position and then dropped on to a recording sheet from a height of approximately 30 cm. This procedure was repeated three times. The distance between two-foot prints was measured and average of the three-foot splay values was calculated.

Grip Strength: Grip strength of both forelimb and hindlimb was measured with a grip strength meter to determine the ability of each rat to grasp and hold on the mesh platform. The grip strength of each rat was measured for 3 consecutive times and the results were averaged separately for the forelimb and hindlimb.

Motor Activity: Motor activity of each rat was monitored using an automated photobeam activity system equipped with a computer analyser. Rats were monitored for three consecutive 10 minutes intervals (total 30 minutes for each rat) allowing for examination of both exploratory and acclimation activity levels. The motor activity parameters i.e., total activity, ambulatory activity and fine activity were evaluated and reported.

Pups Observation:Each litter was examined as soon as possible after delivery to establish the number of pups, sex of pups, stillbirths, live birth, runts, and the presence of gross anomalies. Pups which died during lactation were weighed and subjected to a post-mortem examination. Pups found dead on the day of littering were examined for possible defects and cause of death and discarded in absence of gross findings.
Each pup was observed for the presence of milk in stomach on PND 0 to ensure nursing care.
AGD of each pup was measured on PND 0. Male pups were observed for the retention of nipples/areolae on PND 13.

Pup Bodyweight: Individual pup bodyweight was recorded on PND 0, 4, 7, 14 and at the time of death.

Blood and Uring Collection: At the time of terminal sacrifice, blood (approximately 3.5 mL) was collected from all rats/sex/group (except female sacrificed on GD 25) under anaesthesia (isoflurane) by orbital plexus puncture. Rats were deprived of food overnight (allowed water ad libitum) prior to blood collection. Blood samples were collected for haematology (in vials containing 4% EDTA), coagulation parameters PT and APTT (in vials containing 3.2% sodium citrate), clinical chemistry, and thyroid hormone analysis (in vials without anticoagulant). Samples were collected at approximately the same time per day. At the time of terminal sacrifice, urine samples were collected overnight from five rats (adult)/sex/group in graduated collecting tubes.
Blood was also collected through decapitation for serum thyroid hormone analysis from two surplus pups/litter (wherever available) on postnatal day 4, and two pups/litter on the day of terminal sacrifice i.e., PND 14. Pup blood was pooled by litter. Blood samples were collected in vials without anticoagulant.


Clinical Pathology Observations: The haematology, clinical chemistry, and urine parameters were analysed from a five rats/sex/group.



Thyroid Hormone Analysis : Serum thyroid hormones (T4, TSH, and T3) levels were analysed from parental male rats, parental female rats and terminal sacrificed pups (PND 14). Serum thyroid hormone (T4) level was analysed from PND 4 pups.
Serum thyroid hormones (T3, T4) were analysed at JRF using validated bioanalytical (JRF Study N° 228-2-14-18247) method. Level of TSH in serum was analysed by ELISA methods according to its kit literature.


Sacrifice and pathology:

Necropsy: All rats were sacrificed by using an overdose of CO2. Male rats were sacrificed after approximately 80% of female animals have delivered. Female rats were sacrificed on post partum day 15 and pups were sacrificed on post partum day 14. Females who did not mate were sacrificed on day 25 after the last day of mating period. Females who had not delivered by day 25 post-coitum were sacrificed on post-coitum day 25. Culled pups on PND 4 (not subjected for hormone analysis) were sacrificed through intraperitoneal administration of thiopentone sodium. Rats belonging to recovery groups were sacrificed after 14 days after scheduled sacrificed of dams.
Gross necropsy was conducted under the direct supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. After opening the abdominal cavity, animals were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out blood from the animal. Care was taken to avoid any damage to the visceral organs while opening the body cavities. The thoracic and abdominal cavities were cut, opened and a thorough examination of the organs was carried out to detect abnormalities. Gross examination of rats, including pups was conducted.
At the time of sacrifice, all adult rats and pups (PND 4 and PND 14) were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to external reproductive genitals which were examined for signs of altered development. All live or dead pups were examined externally with particular attention to the external reproductive genitals which were examined for signs of altered development.
The uteri of all cohabited female rats were examined and the number of implantation sites and the number of corpora lutea were recorded.
Pups which were found dead on PND 0 were subjected to gross examination and portion of lung was immersed in water for confirmation of live (stillbirth) or dead status at the time delivery. Pups which were found dead was observed for the presence of a milk band. Pups without gross lesion were discarded after examination.

Organ Weight and Preservation of Organs/Tissues: At the time of sacrifice, organs/ tissues of rats were excised, weighed and fixed (in 10% neutral buffered formalin except for testes which were fixed in Modified Davidson’s fixative) for microscopic examination

Histopathology: Detailed histopathological examination of preserved organs including gross lesions was performed in high and vehicle control dose group animals. Detailed histopathological examination was also performed for other groups in which gross lesions were observed.
Detailed testicular histopathological examination, paraffin embedding and transverse sections of 4-5 µm thickness) was conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation included identification of treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis included the caput, corpus and cauda, which was accomplished by evaluation of a longitudinal section. The epididymis was evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types and phagocytosis of sperm. Periodic Acid Schiff (PAS) and, hematoxylin and eosin staining were used for examination of the testes, while hematoxylin and eosin were used for epididymides and ovaries.
Histopathological examination of the ovary was carried out to detect treatment-related effects such as qualitative depletion/increase of primordial, secondary, antral, graffian follicles population, persistence and increased/decreased corpus luteum, ovarian degeneration/atrophy and stromal cell proliferation.
Statistics:
The statistical analysis was carried out for the parameters using validated software developed at JRF. Non-pregnant rats were excluded from statistical analysis. Assumed pregnant rats were excluded from statistical analysis of gestation phase parameters.
Data such as bodyweight, bodyweight gain, food consumption, hind limb foot splay, grip strength, organ weight, relative organ weight, number of implantation, pre-natal loss, post-natal loss, male sex ratio, estrous cycle length, haematology and clinical chemistry parameters, some urinalysis parameters and litter parameters (pups bodyweight and pups bodyweight gain) were subjected to Shapiro-Wilk’s test for checking normality wherever applicable, followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data did not meet the normality, data was transferred to log form or square root to check the normality again. When log transfer or square root data did not meet the normality, Kruskal-wallis or Mann-whitney test was performed to calculate significance. When the data did not meet the homogeneity of variance, statistical analysis was extended following decision tree as provided by Gad, S.C., 2007. AGD was normalised (the ratio of AGD to the cube root of bodyweight) and then subjected for statistical analysis.
Count data {viz., litter size, number of implant, pre-coital interval, duration of gestation, number of estrous cycle, urination count, defecation count, rearing count, motor activity (total, fine and ambulatory)} were subjected to non-parametric test either Kruskal-wallis test or Mann-whitney test.
Non-parametric data such as gestation index, parturition index, pregnancy rate, survival index, mortality index, live birth index and fertility index were analysed using a Chi-Square test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All rats belonging to control and test item treated groups were normal throughout the study period.
Mortality:
no mortality observed
Description (incidence):
No treatment-related mortality or morbidity was observed during the study period in control and test item treated groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean bodyweight of low, mid and high dose (main and recovery) groups was comparable with respective control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Male rats:The mean food consumption of low, mid and high dose (main and recovery) groups was comparable with that of the respective control group. Statistically, a significant decrease in the food consumption in the low and mid dose groups during treatment days 1-8, 8-15 and 1-42 was observed when compared with respective control group. Statistically significant decrease in food consumption in low dose group during treatment days 29-36 was observed when compared with respective control group. Statistically significant decrease in food consumption in high dose group during treatment days 8-15 was observed when compared with respective control group.
Due to lack of dose-dependency and theother supportive findigs like effects on the bodyweight and bodyweight gain, these could not be considered as the test item related effects.

Female: The mean food consumption of low, mid and high dose (main and recovery) groups was comparable with that of the respective control group. Statistically, a significant decrease in food consumption in the mid and high dose group during gestation days 7-14 was observed when compared with the respective control group. Statistically, a significant decrease in food consumption in high dose group during gestation days 14-20 was observed when compared with the respective control group.
Statistically significant decrease in food consumption in low and high dose group during lactation days 7-14 and 0-14 was observed when compared with the respective control group. Statistically significant decrease in food consumption in high dose group during lactation days 0-4 was observed when compared with respective control group.
Statistically significant decrease in food consumption in recovery high dose group during treatment days 29-36 and 43-50 was observed when compared with respective control group.
Due to lack of the dose dependency and the other supportive findigs like effects on the bodyweight and bodyweight gain, these could not be considered as test item related effects.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Test item treatment did not lead to any alteration in haematological parameters.
In recovery group females (G6), statistically significant decrease was noted in hemoglobin and MCHC as compared to vehicle control recovery group (G5). It was not related to treatment due to absence of effect in terminally sacrificed animals and absence of consistency between sexes.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males, a statistically significant increase in ALT in high dose (G4) as compared to vehicle control group (G1). In absence of histopathological lesions in liver and absence of similar alteration in female animals, this alteration in ALT could not be considered as related to test item treatment.
Additionally, in males, statistically significant decrease in urea and BUN whereas, statistically significant increase in total bilirubin was noted in low dose group (G2). Statisctically significant increase was also noted in bile acids in the low and mid dose group (G2 and G3) as compared to vehicle control group (G1), which could not be considered as treatment related due to absence of dose dependency.
In females of high dose recovery group (G6), a statistically significant increase in ALP, urea, BUN, total bilirubin and bile acids was noted which could not be considered as related to the test item treatment due to the absence of similar findings in terminally sacrificed animals and consistency between sexes.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item treatment did not lead to any alteration in any urinalysis parameters.
Statistically significant decrease in volume was noted in male animals of high dose recovery group (G6), which could not be considered as related to the test item treatment due to absence of similar findings in terminally sacrificed animals and consistency between sexes.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Home Cage Observations
In-home cage, all rats from treatment and control groups revealed normal postures asleep (curled up often asleep), sitting A (sitting but with head hung down), sitting B (sitting normally, feet tucked in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were absent in home cage during NBO.

9.11.2 Handling Observations
NBO performed during handling of rats did not reveal any abnormality related to treatment. All the rats revealed normal behaviour during removal (very easy - animals sit quietly) and handling (easy - alert, limbs put against the body). None of the rats showed lacrimation, salivation or piloerection. Eyelids were wide open in all rats. Eye and skin examination of rats from all groups did not reveal any abnormality.

9.11.3 Open Field Observations
In open field, all rats from treatment and control groups showed normal gait, mobility, arousal and respiration during the two minute observation period. Clonic and tonic movements, stereotypy and bizarre behaviour were absent. No treatment related significant changes were observed in vocalisation, rearing, and urination and defecation counts of male and female rats from treatment groups when compared with the respective control groups.
Some incidental changes in male rats were noted viz., a statistically significant decrease in the rearing count at week 2 in the low dose group and at week 1 in high and recovery high dose group; urination count in high dose group at week 5 when compared with the respective control groups.
Some incidental changes in female rats were noted viz., a statistically significant decrease in rear count at week 3 in the mid dose group and at week 5 in high dose group; defecation count in low dose group at week 1 when compared with the respective control groups.
These changes were considered as incidental in nature based on the absence of dose dependent effect.

9.12 Functional Observational Battery
9.12.1 Motor Activity
The motor activity counts of male and female rats from all treatment groups were comparable with that of the control group. Some incidental changes were observed in female rats like, a statistically significant decrease in fine value at 0-10 minute interval was observed in low dose group when compared with that of the control group. Statistically significant increase in fine value at 11-20 minute interval was observed in recovery high dose group when compared with that of the control group. Statistically significant increase in fine and total value at 21-30 minute interval was observed in recovery high dose group when compared with that of the control group.

9.12.2 Sensory Reactivity Measurements
Sensory reactivity parameters viz., approach response, touch response, click response, pupil response, tail pinch response and air righting reflex in all treatment groups were comparable with that of the control group.

9.12.3 Grip Strength
The hindlimb and forelimb grip strength values of rats from treatment groups were comparable with that of the control group.

9.12.4 Hindlimb Foot Splay
The hindlimb foot splay values of rats from treatment groups were comparable with that of the control group. In recovery female high dose group, a statistically significant increase in foot splay value was observed when compared with that of the control group. This increase could not be considered as test item related effects due to absence of effect on male and female rats (main group) and recovery male rats.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Parents:
Test item treatment did not lead to any alteration in any absolute and relative organ weight parameters.
Statistically significant decrease in organ weight (absolute and relative) of spleen and thyroid with parathyroid was noted in parent male animals of mid dose group (G3) which was not considered as treatment related due to lack of dose dependency.
In females, a statistically significant increase in absolute organ weight of heart was noted in low dose group (G2) as compared to vehicle control group (G1) which was not considered as treatment related due to lack of dose dependency.
Pups:
In male pups, a statistically significant decrease in terminal bodyweight in high dose group (G4) was observed as compared to vehicle control group (G1) which could be considered as treatment related. As the decrease in bodyweight was below 10%, this could be considered as non adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External
External examination of the animals (adult and pups) did not reveal any abnormality.
Internal
Parents: Internal examination of male animals revealed enlarged kidneys (G2: animal N° 29), reduced size of testes, epididymides, seminal vesicles and prostate (G4: animal N° 57), pus forming cavity at cauda epididymides (G5: animal N° 63), reddish foci on duodenum and jejunum (G6: animal N° 68) whereas, female animals revealed mummified fetus in uterus (G1: animal N° 109) and gravid left horn of uteus (G2: animal N° 120). All these macroscopic findings could not be considered as treatment related due to absence of consistency.
Pups: Internal examination of terminally sacrificed pups (PND 4 and PND 14) and found dead pups did not reveal any abnormality of pathological significance.
Neuropathological findings:
no effects observed
Description (incidence and severity):
See behaviour
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related microscopic findings were noted in organ across varoious group (G1 to G6).
Microscopic lesions observed in various organs in male and female animals were at a lower rate of occurrence. Further these lesions were mostly minimal to mild in nature, non-specific, insignificant and correlated to respective gross finding. Hence, these lesions were considered to be spontaneous or incidental and not related to the test item treatment.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No treatment related microscopic findings were noted in organ across varoious group (G1 to G6).
Microscopic lesions observed in various organs in male and female animals were at a lower rate of occurrence. Further these lesions were mostly minimal to mild in nature, non-specific, insignificant and correlated to respective gross finding. Hence, these lesions were considered to be spontaneous or incidental and not related to the test item treatment.
Other effects:
no effects observed
Description (incidence and severity):
MALE, FEMALE AND PUPS
Thyroid hormone i.e., T3, T4 and TSH serum levels were comparable in male, female and pups (PND 4 and 14) when compared with that of the respective control group.

FEMALE RATS:
Evaluation of Estrous Cycle (TABLE 13; APPENDICES 18 and 19)
No effect of test item treatment was observed on the mean number of estrous cycle and mean number of estrous cycle length in low, mid and high dose (Main) group when compared with respective control group.
9.8 Fertility (TABLES 1 and 13; APPENDICES 9 and 17)
Male fertility index, female fertility index, gestation index, parturition index and percentage of pregnant animals were comparable with that of the control group. The duration of the gestation days and pre-coital interval were comparable among all the test item treated groups with that of the control group. Statistically significant decrease in duration of gestation in low dose group was observed when compared with the respective control group.
Due to lack of the dose dependency, this effect could be considered as an incidental in inature.
9.9 Pre and Post-natal Data (TABLE 13; APPENDIX 17)
The Mean number of implants, mean number of pre and post-natal loss, mean percentage of pre and post-natal loss was comparable among all the test item treated groups with that of the control group.




PUPS OBSERVATIONS
9.10.1 Mortality and Mortality Index (TABLE 13; APPENDIX 10)
Statistically significant increase in pup mortality was observed during PND 0 – 4 in female and composite of male and female pups belonging to high dose group when compared with that of the control group.
These significant increase in mortality during PND 0 -4 was due to complete loss of litter of Rat N° 146 belonging to high dose group. This effect could not be considered as the test item related effect.
9.10.2 Clinical Sign (TABLE 13; APPENDIX 10)
All pups belonging to control and test item treated groups were normal throughout the study period.
9.10.3 Bodyweight and Bodyweight Gain (TABLES 11 and 12; APPENDICES 12, 13 and 14)
Statistically significant increase in bodyweight in male, female and composite of male and female pups on PND 14 was observed in mid dose group when compared with that of the control group. Increase in bodyweight is toxicologically insignificant, hence considered as unrelated to treatment. Statistically significant decrease in bodyweight in male, female and composite of male and female pups on PND 14 was observed in high dose group when compared with that of the control group.
Statistically significant decrease in bodyweight gain in male pups during PND 7-14 was observed in high dose group when compared with that of the control group.
These decrease in bodyweight was due to decrease in maternal food consumption during that particular interval and due to lack of dose dependency, these effect could be considered as incidental in inature.
9.10.4 Live Birth and Survival Index (TABLE 13)
Live birth index of test item treated group was comparable with that of the control group. Survival index of high dose group was comparable with that of the control group with exception of statistically significant decrease was observed in female and composite of male and female pups which was considered as unrelated to treatment based on complete loss of litter of Rat N° 146 belonging to high dose group.
9.10.5 Litter Size and Male Sex Ratio (TABLES 10 and 13; APPENDIX 11)
No treatment related differences were observed in mean counts of male pups, female pups, and composite of male and female pups in low, mid and high dose groups (Main) when compared with the respective control group.
The sex ratio of test item treated groups was comparable with that of the control group. 
9.10.6 Ano-genital Distance and Nipple Retention (TABLE 11; APPENDICES 10, 15, and 16)
The ano-genital distance measured on PND 0 of the test item treated groups was comparable with that of the control group. None of the male pups belonging to either control or the test item treated groups showed retention of nipples.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
125 mg/kg bw/day (actual dose received)
System:
other: At 125 mg/kg bw/day no treatment related systemic or local adverse effect was found
Organ:
other: At 125 mg/kg bw/day no treatment related systemic or local adverse effect was found

Applicant's summary and conclusion

Conclusions:
Based on the results of present study, it is concluded that:
- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 125 mg/kg b. wt./day based on no effect on systemic end-points.
- NOAEL for fertility toxicity was 125 mg/kg b. wt./day based on no effect on fertility.
- The NOAEL for reproductive toxicity was at 125 mg/kg b. wt./day dose level based on no treatment related effects on reproduction.
- The NOAEL for developmental toxicity was 125 mg/kg b. wt./day dose level based on no effect on mortality, clinical sign, and, postnatal growth.
Executive summary:

The study was conducted to determine initial information on the toxic characteristics, systemic toxicity, reproduction/developmental toxicity and neurotoxicity whenO-chlorobenzaldehydeis administered by gavage to male and female Wistar rats during pre-mating, mating, gestation, and lactation period.

 Method:

Route of Administration           :

Oral through gavage.

N° of Animals            :

Main groups:15 rats/sex/group.

Recovery groups:5 rats/sex/group.

Frequency of Dose Preparation                 :

 

twice a week.

Frequency of Dose

Formulation Analyses :

 

Once before initiation of treatment and twice during the treatment period.

Dose Levels

(mg/kg b. wt./day)       :

 

Main groups:G1 (control): 0, G2 (low dose): 5, G3 (mid dose): 25, G4 (high dose): 125

Recovery groups:G5 (control): 0; G6 (high dose): 125

Duration of Treatment :

Male: two weeks prior to mating, during the mating (two weeks) and until approximately 80% of the females have delivered. Total days of treatment was 42 days.

Female: two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery.

Recovery male and female: Treatment was given till the first scheduled sacrifice of lactating female rats and then observed further for a period of 14 days without any treatment.

Mortality & Morbidity:

Twice daily.

Clinical Sign               :

Twice during the treatment and recovery period.


 

 

Bodyweight                :

Main groups:

Male: Weekly throughout treatment period and at the sacrifice.

Female: Weekly throughout pre-mating and mating periods.

On Gestation day (GD) - 0, 7, 14 and 20.

On Lactation day (LD) - 0, 4, 7, 14 and 15.

Pups: On Postnatal (PND) - 0, 4, 7 and 14.

Recovery groups:Male and Female groups: Weekly throughtout treatment, recovery period and at the sacrifice.

Food Consmuption      :

Main groups:

Male: Weekly throughout treatment period (except mating period).

Female: Weekly throughout the pre-mating period.

On GD-0, 7, 14 and 20. On LD 0, 4, 7, and 14.

Recovery groups:

Male and Female groups: Weekly throughtout treatment and recovery period.

Estrous Cycle             :

Main groups:

Two weeks pre-treatment, pre-mating period and during the mating period until evidence of mating is observed. Estrous cycle was also observed on the day of terminal sacrifice.

Neurobehavioural Observation (NBO)     :

 

Once before initiation of treatment and weekly throughout treatment and recovery periods.

Functional Battery Observation (FOB)     :

 

From 5 rats/sex/group before terminal and recovery sacrifice.

Pup Observation         :                                   

Clinical sign - once daily. Ano-genital distance (AGD) on PND 0. Nipple retention in male pups on PND 13.

Blood Collection         :

From all adult male rats at TS and lactating female rats on LD 15 through retro-orbital plexus under anaesthesia.

From pups on PND4 (standardised) and 14 through decapitation.

From all recovery male and female rats at TS through retro-orbital plexus under anaesthesia.

Clinical Pathology      :

From 5 rats/sex/group at terminal and recovery sacrifice.

Thyroid Hormone       :

Main groups:

T3, T4 and TSH: From all parent male and female rats.

From PND 4: T4 (litter-wise).

From PND 14: T3, T4 and TSH (litter-wise).


 

 

Sacrifice                     :

Main groups:

Male: Approximately 80% of females delivered

Female: On LD 15; Pups: On PND 14.

Recovery groups:

Male and female: After 14 day of the sacrifice of the first lactating female rat.

Organ Weights            :

Testes, epididymis, levator ani plus bulbocavernosus muscle complex, Cowper’s glands, glans penis, prostate and seminal vesicles with coagulating glands as a whole, paired ovaries (wet weight), adrenals, thyroid with parathyroid and uterus (including oviduct and cervix) from all parental rats. Liver, kidneys, thymus, spleen, brain and heart from 5 rats/sex/group.

Thyroid with parathyroid (post-fixation) from 1 male and 1 female pup/litter.

Histopathology           :

Detailed histological examination of preserved organs was performed in high dose and control group rats. Histopathology was also performed for organs with gross abnormalities.

 

Results:

The results of dose formulation analyses were within the acceptable range of ±15% of the nominal concentration and the %CV was less than 10 which suggests that the prepared dose had an acceptable a.i. concentration and it was homogeneously prepared.

 

O-chlorobenzaldehyde at 5, 25 and 125 mg/kg b. wt./day

-            No signs of toxicity and mortality were observed in male and female rats.

-            No effect on the bodyweight, bodyweight gain and the food consumption was observed in male and     female rats.

-            NBO (Neurobehavioural observations) and FOB (Functional Observational Battery) parameters were comparable to controls.

-           No treatment-related effect on pre-natal loss, percent pre-natal loss, post-natal loss, percent post-natal loss, mating index, male fertility index, female fertility index, gestation index,        parturition index, pre-coital interval, percent of pregnant females, and duration of gestation was       observed.

-             No effect on sex ratio, mean estrous cycle length and the mean number of estrous cycles prior to mating was observed.

-             No effect on litter size (number of pup counts) and number of implants was observed.

-             In pups, no effect on bodyweight, bodyweight gain,and AGD and nipple retention was observed.

-     No treatment-related effect on mortality index, live birth index and survival index was observed.

-     External and internal examination of the animals (all adult animals and pups) did not reveal anylesions.

-            No treatment-related effect on thyroid hormone (T3, T4 and TSH) was observed in male and female    rats, pups of PND-4 and 14.

-            Organ (absolute and relative) weight was comparable with that of the control group.

-            External and internal examination of the animals (all adult animals and pups) did not reveal anylesions. No treatment-related histopathological findings was observed.

 

Conclusion

Based on the results of present study, it is concluded that:

-         No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 125 mg/kg b. wt./day based on no effect on systemic end-points.

-           NOAEL for fertility toxicity was 125 mg/kg b. wt./day based onno effect on fertility.

-           The NOAEL for reproductive toxicity was at 125 mg/kg b. wt./day dose level based on no treatment related effects on reproduction.

-           The NOAEL for developmental toxicity was 125 mg/kg b. wt./day dose level based on no effect on mortality, clinical sign, and, postnatal growth.

COMPLIANCE:Signed and dated GLP and Quality Assurance statements are provided. There was no deviation from regulatory requirements.