Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2019 to February 2020
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-bis[[bis(2-hydroxyethyl)amino]methyl]-4-nonylphenol
EC Number:
243-500-6
EC Name:
2,6-bis[[bis(2-hydroxyethyl)amino]methyl]-4-nonylphenol
Cas Number:
20073-51-2
Molecular formula:
C25H46N2O5
IUPAC Name:
2,6-bis[[bis(2-hydroxyethyl)amino]methyl]-4-nonylphenol
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Dose formulations and vehicle were administered to male and female rats daily up to and including the day before scheduled sacrifice. The dose-volume for administration was 10 mL/kg body weight. The dose-volume was adjusted according to the most recently recorded body weight. The control group was received vehicle alone. The first day of dosing was designated as day 1 for all rats.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The active ingredient (a.i.) concentration and homogeneity of the test item were analysed once before initiation of treatment and twice during the treatment period. Two sets of samples (10 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from each concentration except control (only one aliquot). One set of samples was used for analyses and the other set of samples was stored at 2 - 8 °C. On each occasion, the mean concentration was determined and compared with the nominal value. The acceptance criteria were ± 15% deviation from nominal value and %CV < 10. The samples were analysed at JRF using a validated analytical method (JRF Study N° 228-2-13-21964). Results are appended in the study report (APPENDIX 35). Stored samples will be disposed of upon finalisation of the report.
Duration of treatment / exposure:
Dosing of both sexes was initiated 2 weeks prior to the mating and continued during the mating period. After mating, the male rats were further dosed up to and including the day before scheduled sacrifice. Female rats were dosed during pregnancy and up to post-partum day 14.
Rats belonging to recovery groups were kept for 14 days after the first scheduled sacrifice of dams, without treatment.
Frequency of treatment:
Dose formulations and 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 (nominal)
Remarks:
Control Group
Dose / conc.:
37.5 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
high dose
No. of animals per sex per dose:
Group N° Group Dose
(mg/kg b. wt./day) Rat N° Sex N° of Rats/Sex
From To
Main Groups
G1 Control 0 1 15 Male 15
16 30 Female 15
G2 Low Dose 37.5 31 45 Male 15
46 60 Female 15
G3 Mid Dose 75 61 75 Male 15
76 90 Female 15
G4 High Dose 150 91 105 Male 15
106 120 Female 15
Recovery Groups
G5 Control 0 121 125 Male 5
126 130 Female 5
G6 High Dose 150 131 135 Male 5
136 140 Female 5
Control animals:
yes, concurrent vehicle
Positive control:
No

Examinations

Observations and examinations performed and frequency:
OBSERVATIONS
7.1 Mortality and Morbidity
Rats were observed daily, twice, for mortality and morbidity.
7.2 Clinical Sign
Rats were observed daily twice, during treatment and recovery periods, for any visible clinical signs.
7.3 Body Weight
Body weight of all-male rats was recorded on the first day of dosing and at weekly intervals thereafter.
Body weight of all female rats was recorded on the first day of dosing and at weekly intervals during the pre-mating period. During the gestation period, female rats were weighed on gestation day 0, 7, 14, 20, and 25. During the lactation period, female animals were weighed within 24 hours of parturition (day ‘0’ post-partum/lactation day), and on post-partum days 4, 7, and 14. Parturition day ‘0’ was defined as the day on which the female littered.
Body weight of all rats belonging to recovery groups was recorded on the first day of dosing and at weekly intervals thereafter.
On the day of fasting, body weights of all surviving rats were recorded. Body weights of all rats were also recorded on the day of sacrifice.
7.4 Food Consumption
The food consumption was determined by differentiating the weight of food input and leftover.
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 day 0, 7, 14, and 20. During the lactation period, food weights were measured on day 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.
7.5 Oestrous Cycle
Oestrous cycle length and pattern were evaluated by vaginal smears observation 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 rat, was also observed on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.
7.6 Neurobehavioral 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. As per JRF/TOX/SOP-375 (Issue I), on day of Functional battery observation (FOB), NBO was performed before the start of FOB.
7.6.1 Home Cage Observation
In-home cage, rats were observed for posture and presence or absence of convulsions as mentioned below:
Text Table 3: 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)

Parameters Observations
Clonic Movement Present
Absent
Tonic Movement Present
Absent
7.6.2 Handling Observation
After completion of home cage observations, each rat was picked up by the observer and observed for below-mentioned parameters:
Text Table 4: Handling observation
Parameters Observations
Ease of removing the animal from the 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)
Parameters Observations
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 mucous membrane)
Chemosis (swelling of the conjunctiva)
Cataract (opacity of the lens)
Corneal opacity (an opaque spot or area on the cornea)
Microphthalmos (abnormally small eyeball)
Exophthalmos (abnormal protrusion of the eyeball)
Piloerection Absent
Present
Skin examination Alopecia (absence or loss of hair)
Rough coat (ungroomed or greasy hair coat)
Dermatitis (inflammation of the 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)

7.6.3 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 2 minutes. During the 2 minutes period, each rat was observed for the below-mentioned parameters:

Text Table 5: Open field observation
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 the 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 backwards)
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 the faecal bolus in open field)
Stereotypy Absent
Circling
Excessive grooming
Other (actual observations)
Bizarre behaviour Other (actual observations)
Self-destructive biting (e.g., tail, paws) or self-mutilation
Biting of open field/standard arena
Absent
Retropulsion (moving backwards)


7.7 Functional Observational Battery (FOB)
The below mentioned FOB parameters along with NBO parameters were performed from randomly selected at least five rats/sex/group towards the end of the study.
7.7.1 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.

7.7.2 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 the average of the three-foot splay values was calculated.
7.7.3 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.
7.7.4 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.
7.8 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 the absence of gross findings.
Each pup was observed for the presence of milk in the 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.
7.8.1 Pup Body Weight
Individual pup body weight was recorded on PND 0, 4, 7, and 14.
7.9 Blood and Urin Collection
At the time of terminal sacrifice, blood (approximately 3.5 mL) was collected from all surviving rats/sex/group 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 and finished by 1:00 PM.
At the time of terminal sacrifice, urine samples were collected overnight from five rats (adult)/sex/group in graduated collecting tubes.
Blood was also be 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.
7.10 Clinical Pathology Observations
The following haematology, clinical chemistry, and urine parameters were analysed from a minimum of five rats/sex/group.
Text Table 6: Haematology parameters
Parameter Parameter
Haematocrit Platelets Count
Haemoglobin Erythrocyte Count (Red Blood Cells)
Mean Corpuscular Haemoglobin Differential Leukocyte Count
Mean Corpuscular Haemoglobin Concentration Reticulocyte Count
Mean Corpuscular Volume Prothrombin Time (PT)
Total Leukocyte Count (White Blood Cells) Activated Partial Thromboplastin Time (APTT)

Text Table 7: Clinical Chemistry Parameters
Parameter Parameter
Alanine Aminotransferase Total Cholesterol
Albumin Total Protein
Alkaline Phosphatase Total Bilirubin
Aspartate Aminotransferase Triglycerides
Creatinine Blood Urea Nitrogen
Creatine Kinase Calcium
Gamma Glutamyl Transpeptidase Albumin : Globulin Ratio
Glucose Potassium
Globulin Sodium
Inorganic Phosphorous Chloride
Lactate Dehydrogenase Urea
Bile Acids -


Text Table 8: Urinalysis Parameters
Physical Observation
Chemical Observation*
Appearance
Colour
Volume [mL]
Specific Gravity
pH
Protein
Glucose
Ketones
Blood/blood Cells
Bilirubin
Urobilinogen [EU/dL]
Keys: * = Chemical parameters will be analysed using a Urine Chemistry Analyzer (Clinitek- Status®)
7.11 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 also analysed from PND 4 pups.
Serum thyroid hormones (T3, T4) were analysed at JRF using validated bioanalytical method (JRF Study N° 228-2-14-18247). Level of TSH in serum was analysed by ELISA methods according to its kit literature. Any residual/retained serum sample will be discarded after the issue of the final report.
Text Table 9: N° of samples collected from parent rats
Group N° N° of Rats/Group Thyroid Hormone
T3 T4 TSH
Male
G1 to G4 15 √ √ √
Female
G1 10 √ √ √
G2 10 √ √ √
G3 6 (T3), 7(T4) √ √ √
G4 11 √ √ √
Key: √ = Activity performed
Text Table 10: N° of samples collected from PND4 and 14 pups – litter-wise
Group
N° N° of Litter/Group – PND 4 Thyroid Hormone N° of Litter/Group – PND 14 Thyroid Hormone
T4 T3 T4 TSH
G1 4 √ 10 √ √ √
G2 4 √ 10 √ √ √
G3 6 √ 8 √ √ √
G4 2 √ 5 √ √ √
Key: √ = Activity performed
7.12 Necropsy
Surviving rats (including pups of PND 14) were sacrificed by carbon dioxide asphyxiation. Male rats were sacrificed after 65% of females have delivered. Culled pups on PND 4, which were not subjected for blood collection, were sacrificed through intraperitoneal administration of thiopentone sodium. Pups were sacrificed on PND 14. Female rats were sacrificed on LD 15. Female rats which did not deliver by day 25 post-coitum were sacrificed on post-coitum day 25. Rats belonging to recovery groups will be sacrificed after 15 days after the first scheduled sacrifice 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, rats were exsanguinated by cutting the abdominal aorta or posterior vena cava to drain out the blood from the rat. 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 organs was carried out to detect abnormalities. Special attention was paid to organs of the reproductive system.
At the time of sacrifice or death during the study, all adult rats and pups were examined macroscopically for any structural abnormalities or pathological changes. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development.
The uteri of all cohabited female rats were examined for the presence and number of implantation sites. The number of corpora lutea was recorded from those female rats which are sacrificed on gestation day 25 and implants were observed.
Pups which were found dead on PND 0 were subjected for gross examination and portion of lung was immersed in water for confirmation of live and dead status at the time of delivery (stillbirth or dead). Pups which were found dead were observed for the presence of milk band. Pups without gross lesions were discarded after the examination.
7.13 Organ Weight and Preservation of Organs/Tissues
At the time of sacrifice, the following organs/tissues of rats were excised, weighed, and fixed in an appropriate fixative to permit microscopic examination:

Text Table 11: Organ weight and preservation
Organ/Tissues Weighed From
Testes * All adult rats

Epididymis *
Levator ani plus bulbocavernosus muscle complex (LABC) *
Cowper’s glands *
Glans penis *
Ovary *
Thyroid gland *
Seminal vesicles with Coagulating gland *
Prostate *
Uterus with oviducts and cervix *
Gross lesions -
Vagina -
Male mammary gland -
Pituitary -
Adrenals *

Organ/Tissues
Weighed From
Liver * 5 adult rats/sex/group

Kidneys *
Thymus *
Spleen *
Brain
(cerebrum, cerebellum, and medulla/pons) *
Heart *
Spinal cord
(at three levels; cervical, mid-thoracic and lumbar) -
Stomach -
Small intestine (Duodenum, jejunum, ileum) -
Large intestine (Caecum, colon, rectum) -
Eye -
Trachea -
Lungs (preserved by inflation with fixative and then immersion) -
Urinary bladder -
Lymph nodes (mandibular, mesenteric) -
Sciatic nerve -
Skeletal muscle and bone -
Bone marrow (Femur) -
Keys: * = Weigh and preserve, - = Preserve
The thyroid gland was excised, collected, weighed and preserved from one male pup and one female pup (wherever available)/litter on the day of terminal sacrifice.

7.14 Histopathology
A detailed histopathological examination of preserved organs including gross lesions was performed in high and control dose group rats.
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), haematoxylin, and eosin staining were used for examination of the testes, while haematoxylin 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.
In addition, all gross lesion as well organs which showed treatment-related microscopic finding (stomach, kidneys, bone marrow femur and spleen) were processed from all lower dose groups and all recovery group and examined microscopically.

Sacrifice and pathology:
Main groups:
Male: Approximately 65% of female rats delivered
Female: On LD 15; Pups: On PND 14. Recovery groups:
Male and female: After 15 days from the sacrifice of the first lactating female rat.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation (mild to moderate) was observed approximately 3 to 5 minutes after dosing in all male and female rats of all test item treated groups and persisted for approximately 20 to 25 minutes. This finding is considered a response to the dose solution and toxicologically non-adverse in nature.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No treatment-related mortality or morbidity was observed during the study period in the control and test item treated groups.
One female rat (Rat N° 108) was found dead on treatment day 10. Necropsy findings in this rat (reddish discolouration in lungs during gross observation and haemorrhages and granuloma during histological examination) suggest that this death was due to gavage accident.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body Weight (TABLE 3 and 4; FIGURE 1, 2, 11, 12, 21, and 26; APPENDIX 3 and 4)
Male:
Marginal decrease in mean body weight of male rats, belonging to 150 mg/kg b. wt./day dose group (main and recovery), was observed.
The mean body weight of male rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups was comparable with that of the control group.

Female:
Marginal decrease in mean body weight of female rats, belonging to 150 mg/kg b. wt./day dose group was observed during the pre-mating period.
Marginal decrease in mean body weight of female rats of recovery group, belonging to 150 mg/kg b. wt./day dose group was also observed.
During gestation and lactation periods, the mean body weight of female rats belonging to 150 mg/kg b. wt./day dose groups was comparable with that of the control group.
The mean body weight of female rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was comparable with that of the control group during pre-mating, gestation and lactation periods except for a statistically significant decrease in the mean body weight of female rats, belonging to 75 mg/kg b. wt./day dose group, on lactation day 14.
9.5 Body Weight Gain (TABLE 5 and 6; FIGURE 3, 4, 5, 6, 13, 14, 15, 16, 22, 23, 27, and 28; APPENDIX 5 and 6)
Male:
Statistically significant decrease in mean body weight gain of male rats, belonging to 150 mg/kg b. wt./day dose group was observed during treatment days 8-15, 29-26 (main and recovery), 36-43 (recovery), 1-50 and 1-53 (recovery) when compared with that of the control group. The mean body weight gain of male rats, belonging to 150 mg/kg b. wt./day dose group was also decreased during treatment days 1-8, 15-22, and 43-50 without statistical significance.
Mean body weight gain of male rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was comparable with that of the control group except for a statistically significant increase in mean body weight gain of female rats, belonging to 37.5 and 75 mg/kg b. wt./day dose group, during treatment days 36-43.
Female:
Statistically significant decrease in mean body weight gain of female rats, belonging to 150 mg/kg b. wt./day dose group, was observed during pre-mating days 1-15 when compared with that of the control group. The mean body weight gain of female rats, belonging to 150 mg/kg b. wt./day dose group, was also decreased during pre-mating days 1-8 and 8-15 without statistical significance.
Marginal decrease in mean body weight gain of female rats of recovery group, belonging to 150 mg/kg b. wt./day dose group, was also observed except a statistically significant increase in mean body weight gain of female rats during treatment days 36-43 and 50-53.
Mean body weight gain of female rats, belonging to 150 mg/kg b. wt./day dose groups was comparable with that of the control group during the gestation and lactation period.
During pre-mating, gestation, and lactation periods, mean body weight gain of female rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was comparable with that of the control group except for a statistically significant decrease in mean body weight gain of female rats, belonging to 37.5 mg/kg b. wt./day dose group, during pre-mating days 1-15.
Marginal decrease in mean body weight and body weight gain in male and female (pre-mating period) rats are correlated with decreased mean food consumption and considered as effects of test item but non-adverse in nature.
Body Weight and Body Weight Gain
The mean body weight and body weight gain of male, female and composite of male and female pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day dose groups were comparable with that of the control group.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Male
Statistically significant decrease in mean food consumption of male rats, belonging to 150 mg/kg b. wt./day dose group was observed during treatment days 1-8 (main and recovery), 8-15, 29-36, 36-43, 43-50, and 1-50 when compared with that of the control group.
Mean food consumption of male rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups was comparable with that of the control group.
Female
Statistically significant decrease in mean food consumption of female rats, belonging to 150 mg/kg b. wt./day dose group, was observed during pre-mating days 1-8 (main and recovery), 8-15, and 1-15 when compared with that of the control group.
During the gestation period, the mean food consumption of female rats was comparable with that of the control group.
Statistically significant decrease in mean food consumption of female rats, belonging to 150 mg/kg b. wt./day dose group was observed during lactation days 0-4, 7-14, and 0-14 when compared with that of the control group. In the absence of supporting findings in lactation body weight and body weight gain, decreased mean food consumption during lactation period is considered as incidental.
During pre-mating, gestation, and lactation periods, mean food consumption of female rats, belonging to 37.5 and 75 mg/kg b. wt./day dose groups, was comparable with that of the control group except for a statistically significant decrease in mean food consumption of female rats, belonging to 37.5 mg/kg b. wt./day dose group, during pre-mating days 8-15.
A decrease in the mean food consumption of male and female (pre-mating) rats was considered as the effect of the test item treatment but non-adverse in nature.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In male rats of high dose group, statistically significant increase in WBC, neutrophil, monocyte and platelets was observed, while statistically significant decrease in haemoglobin, haematocrit, MCV and MCH was noted. In female rats of high dose group, statistically significant decrease in RBC, while statistically significant increase in lymphocytes was noted. These effects were related to test item treatment as it was well correlated with inflammatory reaction observed in stomach.
After 14-days of recovery period, statistically significant decrease in MCH and statistically significant increase in reticulocytes was noted in male rats of high dose recovery group. Increase in reticulocytes was due to decrease in haemoglobin and haematocrit at terminal sacrifices. Results shows that effects were recovered almost completely after 14-days of recovery period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In high dose male rats, statistically significant decrease in albumin was noted, while in high dose female rats decrease in albumin was observed without statistical significance. Statistically significant decrese in albumin: globulin ratio was noted in both sexes of high dose group. It was related to decrease in albumin. Effects were considered as related to test item treatment and were recovered after 14-days of recovery period.
In high dose group, statistically significant decrease was noted BUN and urea in females. Effect was less likely to be related to treatment as it was not observed in males.
Statistically significant decreases in LDH and CK in high dose male rats and in ALT in mid dose male rats were noted. Similarly, in treated recovery group, statistically significant decrease was noted in ALT in both sexes. These effects were considered as toxicologically insignificant.
Statistically significant decrease was observed in glucose in low dose and high dose female rats and increase in AST in low dose female rats. These effects were unrelated to test item treatment due to lack of dose dependency and consistency between sexes.
In treated recovery group male rats, statistically significant decrease and increase was noted in creatinine and phosphorus, respectively. It was unrelated to test item treatment in absence of similar effects at the end of treatment.
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 increase in specific gravity was noted in G6 females, which was not considered as related to 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):
Neurobehavioral Observations
9.11.1 Home Cage Observations (TABLE 13 and 14; APPENDIX 21 and 22)
In the 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 the home cage during NBO.
9.11.2 Handling Observations (TABLE 13 and 14; APPENDIX 21 and 22)
NBO performed during handling of rats did not reveal any abnormality related to treatment. All the rats revealed a 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 (TABLE 13 and 14; APPENDIX 21 and 22)
In the open field, all rats from treatment and control groups showed normal gait, mobility, arousal and respiration during the two minutes observation period. Clonic and tonic movements, stereotypy and bizarre behaviour were absent. No treatment-related significant changes were observed in vocalisation, rearing, urination, and defecation counts of male and female rats from treatment groups when compared with that of the control group.
Some incidental changes were observed in male rats of high dose group (statistically significant decrease in rearing count at week 5 (main and recovery groups) and urination count at week 2) and low dose group (statistically significant increase in rearing count at week 2).
These changes were considered incidental in the absence of other supporting findings.
9.12 Functional Observational Battery
9.12.1 Motor Activity (TABLE 15; APPENDIX 23 and 24)
The motor activity counts of male and female rats from all treatment groups were comparable with that of the control group except statistically significant increase in fine activity of female rats of high dose recovery group for duration of 21-30 minutes.
9.12.2 Sensory Reactivity Measurements (TABLE 16; APPENDIX 25 and 26)
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 (TABLE 17; APPENDIX 27 and 28)
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 (TABLE 18; APPENDIX 29)
The hindlimb foot splay values of rats from treatment groups were comparable with that of the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Terminal Body Weight, Organ Weight and Relative Organ Weight
Parents:
Test item treatment led to a marginal decrease in terminal body weight of high dose male rats without statistical significance. Statistically significant increase in the absolute and relative weight of kidneys was noted in both sexes of high dose group. This effect was related to tubular hypertrophy and considered as related to test item treatment. This effect was also noted in treated recovery group except absolute weight in males.
A statistically significant decrease was noted in absolute weight of prostate + seminal vesicles with coagulating glands in high dose male rats. It was considered as non-adverse as no effect was found in the histopathology.
Statistically significant increase was noted in relative weight of spleen and testes in high dose males, and adrenals in high dose males and females. These effects were more likely to be owed to marginal decrease in terminal body weight. Statistically significant increase was observed in relative weight of adrenal in high dose male, this effect was not considered as related to test item treatment due lack of effect in absolute weight and lack of any microscopic changes.
Statistically significant increase was noted in relative and absolute weight of ovaries in high dose recovery group (G6), which was not considered as related to test item treatment.
Pups:
In male pups, a statistically significant increase was noted in the relative weight of thyroid gland in the high dose group (G4). It was considered as unrelated to test item treatment due to lack of effect in absolute weight and lack of consistency between sexes.
Significant decrease was noted in weight of thyroid gland in mid dose (G3) female was not related to test item treatment in absence of dose dependency.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic Pathological Findings
Parental rat:
External examination of rats of either sex across various groups (G1 to G4) did not reveal any abnormality. Internal examination of adult rats revealed whitish foci in non-glandular stomach in all test item treated main groups except low dose female rats. After 14-days of recovery period marked reduction in severity was observed. The effect was related to the test item treatment as supported by histopathological lesions.
Other lesions noted on gross examination (reduced size of testes, stones in the urinary bladder, and reddish discolouration in lungs) were considered as spontaneous/incidental.
Pups:
External and internal examination of pups (found dead and sacrificed terminally at PND 4 and 14) did not reveal any abnormality.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment of the test item was associated with histopathological lesions (ulceration, hyperplasia, hyperkeratosis and inflammatory cells) in the stomach at all 3 dose levels in dose-dependent manner. After 14-days of the recovery period, though some of the lesions were continued, marked recovery was observed in incidence and severity.
Lesions noted in bone marrow (increased cellularity) and spleen (EMH) in -mid-dose and high dose were considered as related to treatment. These lesions were more likely to be owed to inflammatory lesions in the stomach (supported by haematology and lesion in the stomach). Lesions were recovered completely after the recovery period.
Similarly, treatment also caused tubular hypertrophy in kidneys at all dose levels. Marginal recovery was observed after 14-days of the recovery period.
Microscopic lesions observed in other organs were at a lower rate of occurrence and with minimal to mild severity with an almost similar incidence between control and high dose group. At instances, though differences observed in the incidence of lesions between treated and control group, those lesions were considered as spontaneous or incidental in nature representing the normal physiological/metabolic or congenital changes and not treatment-related.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Live Birth and Survival Index (TABLE 12)
Live birth index of male, female, and composite of male and female pups, belonging to 150 mg/kg b. wt./day dose group was statistically significant decrease when compared with that of the control group.
Survival index of male, female, and composite of male and female pups, belonging to 150 mg/kg b. wt./day dose group was statistically and significantly decreased during the postnatal days 0-4 when compared with that of the control group.
This decreased in the live birth index and survival index were considered as adverse effects of the test item treatment.
Live birth index and survival index of male, female and composite of male and female pups, belonging to 37.5 and 75 mg/kg b. wt./day dose group were comparable with that of the control group.
Litter Size and Male Sex Ratio (TABLE 9; APPENDIX 12)
Male sex ratio and the mean counts of male pups, female pups, and a composite of male and female pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day dose group were comparable with that of the control group.
Ano-genital Distance and Nipple Retention (TABLE 10; APPENDIX 16 and 17)
The anogenital distance of male and female pups, belonging to 37.5, 75, and 150 mg/kg b. wt./day dose 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.
Evaluation of Oestrous Cycle
No effect of the test item treatment was observed on the mean oestrous cycle length and the mean number of oestrous cycles.
Fertility
Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats and mating index were comparable with that of the control group. The duration of the gestation and pre-coital interval was also comparable with that of the control group.

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
ca. 37.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
37.5 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
37.5 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Critical effects observed:
yes
Lowest effective dose / conc.:
37.5 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
other: bone marrow
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

The alterations in the bone marrow are suggested to be a consequence with the inflammatory process ocurring in the stomach

Applicant's summary and conclusion

Conclusions:
Test item treatment produced whitish foci in the non-glandular stomach, histopathological lesions (ulceration, hyperplasia, hyperkeratosis and inflammatory cells) in the stomach, and tubular hypertrophy in kidneys at all three dose levels. Based on these findings, stomach and kidney were identified as target organs and No Observed Adverse Effect Level (NOAEL) for parental toxicity was established below 37.5 mg/kg b. wt./day.
Executive summary:

This study was conducted to determine the initial information on the toxic characteristics, systemic toxicity, reproduction/developmental toxicity, and neurotoxicity when POLIOL MB 600 is administered orally in male and female Wistar rats, through gavage, during pre-mating, mating, gestation, and lactation period.