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Administrative data

Description of key information

Repeated Dose (Oral) Toxicity Study:


In the OECD 422 study with the substance (rats; oral gavage), NOAEL for systemic toxicity was determined to be 1000 mg/kg bw/day in both genders. The study was performed according to GLP and was considered to be reliable without restrictions (Klimisch 1).


 


Repeated Dose (Dermal) Toxicity Study:


A short-term dermal toxicity study does not need to be conducted because exposure of humans via dermal route in production and/or use is unlikely based on the provided thorough and rigorous exposure assessment. Also, the acute dermal toxicity value for test chemical (as provided in section 7.2.3) is >2000 mg/kg body weight. Given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Therefore, it is expected that test chemical shall not exhibit toxicity via dermal route following exposure for 28 days. In addition, there is no data available that suggests that test chemical shall exhibit repeated dose toxicity by the dermal route and therefore this end point was considered for waiver.


 


Repeated Dose (Inhalation) Toxicity:


A short-term inhalation toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment. Also, the given test chemical has very low vapor pressure, < 0.1333 Pa at 20°C, so the potential for the generation of inhalable vapor is very low. The normal conditions of use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be highly unlikely and therefore the end point of repeated inhalation toxicity was considered for waiver.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2022 - Mar 2023
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the recommended species to be used in OECD Test Guideline 422 and this species was also requested to be used by the regulatory authority. The Wistar strain was selected based on available historical control data at the test facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMALS:
Source: Hylasco Biotechnology (India) Pvt. Ltd., CPCSEA Reg.No.1808/P/RcBt/ /15/CPCSEA, Hyderabad500 078, India.
Age at the first day of dosing: 14-15 weeks
Body weight at the first day of dosing: 459-529 g (males) and 244-298 g (females).

ANIMAL HUSBANDRY
Acclimatization: The animals were acclimatized for 9 days to experimental room conditions, before commencement of treatment. Veterinarian examinations for general health were performed on the day of receipt and prior to start of treatment. All animals were found to be healthy and suitable for study inclusion.

Environmental conditions: Animals were housed in environmentally monitored air-conditioned room with adequate fresh air supply (air-change each 12 to 14 hours). The room temperatures ranged from 19.3 to 23.8°C, with relative humidity that ranged from 46 to 70%, and with 12 hours light and 12 hours dark cycles. Room temperatures and humidities were monitored round the clock and recorded once daily.

Housing: Animals of the same sex and group were housed in two and three per cage in standard polycarbonate cages (Size: L 421 X B 290 X H 190 mm). During mating, one male and one female were housed together until conformation of mating. Pregnant females were separated from males and housed individually until sacrifice along with the litters. Enrichment/nesting materials were provided in the dam cages from the day of advanced gestation (GD 18). The cages were fitted with stainless steel mesh top grill with facilities for holding pelleted feed and drinking water bottle.

Water: Reverse osmosis water was available ad libitum throughout the study period. The test results (analytical and bacteriological test) of collected water samples met acceptance criteria as per IS 10500:2012 for drinking water.

Feed: The pellet feed was available ad libitum throughout acclimatization and experimental period. HYLASCO (Batch/Lot. 25MAR20223 and 25JUN20221) manufactured by PMI Nutrition International, US was provided. The analytical results of collected feed samples met acceptance criteria as per FSSC 22000.

Bedding: Autoclaved corncob (Lot No.13 and 14) was used as bedding material and was changed along with the cage at least once a week. The material was manufactured by Rowan Agro Nature Pvt Ltd. The corncob samples passed the requirement of Food Safety Standard Regulation (2011) specified with respect to tested parameters.
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Corn oil was selected as vehicle based on the results of a solubility test performed in the test facility and after consultation with the Sponsor.
Details on oral exposure:
Dose administration was performed at approximately the same time each day for all animals (within ±2 hours) throughout the treatment period. The dose volume administered to each rat was at an equivolume of 5 mL/kg bw throughout the study. The dose volumes were adjusted based on the most recently recorded body weight of the individual rat.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analyses for concentration and homogeneity verification were performed during the first week of pre-mating and during the last week of lactation periods using a validated HPLC method. Concentrations of the top, mid and bottom layers of all dose formulation samples were within the acceptable range of ±15 % to the nominal concentration (from 97.12% to 106.69%) with less than 10% relative standard deviations (from 0.78% to 6.25%). Therefore, all dose formulations prepared in this study were considered to be accurate, precise and homogeneous.
Duration of treatment / exposure:
Males: Male rats of G1, G2, G3 and G4 were treated two weeks before mating and two weeks during the mating period, for a total of 28 treatment days. These animals were sacrificed on the day after the final day of treatment.

Females: Female rats in G1, G2, G3 and G4 were dosed before mating (two weeks; with the objective of covering at least two complete oestrous cycles), two weeks during the mating period, the gestation period, and up to and including post-natal day (PND) 13 (i.e. up to the day before scheduled sacrifice on PND 14), for a total of approx. 58 days.
Frequency of treatment:
Once daily
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
G4
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
G3
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
G2
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
G1 (vehicle control)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Animals: 12 female animals per each main group were examined during the pre-treatment period (14 days) for oestrous cycle evaluation. Females that failed to exhibit typical 4 - 5 days oestrus cycles were not included in the main study. After the exclusion/randomization, 10 female rats were selected for each main group.

Randomization: On the day before commencement of treatment, animals were grouped and allocated to their respective treatment groups using body weight based stratified randomization in MS Excel. It was ensured that the mean body weights of each group before the start of the treatment did not exceed ±20% of the mean body weight in each sex and group. Body weights of the animals were analyzed statistically to rule out any statistically significant differences between groups of same sex.

Justification for dose levels: Dose levels were selected based on the results of a dose range finding (DRF) study performed at the test facility (VBPL-TOX-099-NG-U-2021). In the DRF, 4 rats/sex/dose were treated at 0, 500 and 1000 mg/kg bw/day. Male rats were treated before mating (2 weeks) and during mating (2 weeks) for a total of 28 days. Female rats were treated before mating (2 weeks), during mating (2 weeks), during gestation, and up to day 4 of lactation. No significant signs of toxicity were observed at 500 mg/kg bw/day. At 1000 mg/kg bw/day, dullness was observed in one male rat (from day 4 to 14) and dullness with occasional piloerection was observed in another male rat (from day 11 and throughout the experimental period). Based on the results from the DRF, a top dose of 1000 mg/kg bw/day was selected for the main study. The doses of 0, 100, 300 and 1000 mg/kg bw/day in the main study were mutually agreed upon by the Sponsor and the Test Facility.
Positive control:
None
Observations and examinations performed and frequency:
MORBIDITY AND MORTALITY
The animals were observed for morbidity/mortality twice daily (morning and afternoon or evening) during the experimental period.

CLINICAL SIGNS OF TOXICITY
Animals were observed for clinical signs once per day during the acclimatization period. On the first three days of dosing, post dose observations were recorded twice at approximately 1-hour intervals in the first four hours. Subsequent post-dose observations were recorded approximately one-hour post dose. Special emphasis was given to pregnant female animals. Once before the first exposure and once a week thereafter, detailed clinical observations were made in all parental animals. These observations were made outside the home cage in a standard arena and preferably at the same time of each occasion. Observations included 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), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHTS
Individual body weights of adult males and females were recorded on day 1 of treatment, weekly thereafter, and on the day of sacrifice. During pregnancy, female body weights were recorded on GD 0, 7, 14 and 20 and within 24 hours of parturition (PND 0) and on PND 4 and 13, and on the day of sacrifice.

FEED CONSUMPTION
Feed consumptions were measured once a week during the pre-mating period and on gestation day (GD) 0, 7, 14, and 20 and on lactation day (LD) 0, 6, and 13. The weight of feed provided, and that leftover, were recorded for each cage and the data was used to calculate mean feed consumption by each animal (gram/rat).

OESTROUS CYCLE
Vaginal smears were collected daily during the 2-week premating period to monitor oestrous cyclicity and to select females with normal cycle length (i.e. 4 to 5 days per pattern) for study inclusion. Oestrous cyclicity was monitored by vaginal smears during the mating period until there was evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid damage to mucosa, which can induce pseudo pregnancy.

THYROID HORMONE ANALYSIS
Blood samples for thyroid hormone analysis were collected from all adult animals at terminal sacrifice. Aliquots of serum samples were stored at -70°C or colder until analysis. Serum samples from all adult males were measured for thyroid hormones (T4 and TSH). Serum T4 (Thyroxine) and TSH (Thyroid stimulating hormone) levels were measured using sandwich ELISA and competitive ELISA respectively, with commercially available reagents/kits from KRISHGEN Bio Systems, India.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
The following neurological examinations were conducted for randomly selected 5 males and 5 females in each main group towards the end of the dosing period (last week). The FOB included the following:
a) Home Cage Observations
Each rat was observed in the home cage for activity level and scored.
b) Handheld Observations
Observations were made while holding the subject animal. Animals were observed for lacrimation and salivation, hair coat characteristics (colour/staining, alopecia, and piloerection), degree of eyelid closure, ocular abnormalities and muscle tone or mass. Each observation was recorded using a scoring/ranking system.
c) Open Field Observation
Each rat was placed in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each rat. During this observation period, each rat was evaluated as it moved freely/unperturbed, and the following parameters were recorded using a scoring/ranking system:
• Activity (locomotion).
• Rearings for a standard time.
• Observation of reactivity and arousal.
• Observation of gait and postural characteristics.
• Observation of involuntary/abnormal motor movements (tremors, twitches, clonic
convulsions, tonic convulsions, Stereotypic behavior and bizarre behavior).
d) Sensory Reactivity Measurements
While the rat was in the open field arena and after two minutes of observation, the following sensory reactivity measurements were performed, and the observations were recorded using a scoring/ranking system:
• Approach response
• Touch response
• Click response
• Tail-pinch response
• Pupil response
• Aerial righting reflex
e) Landing Hind Limbs Foot splay
The landing hind limbs foot splay was performed by dropping each rat on a horizontal surface (e.g., table surface) from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was dropped from a height of approximately 30 cm on to a recording paper sheet, which had the details viz., Study number., animal number and group number. A clean recording paper sheet was used for each rat. A total of three readings were made for each rat and the average of three individual foot splay values were presented in the report.
f) Grip Strength Performance
Forelimbs and hind limbs grip strength performance was measured using a grip strength meter (Orchid Scientific & Innovative India Pvt. Ltd; Model: GSM 02RS). Three trials for both fore and hind limb was conducted for each rat. The average value (forelimb and hind limb, respectively) was calculated and presented in the report.
g) Motor Activity
Locomotor activity was tested for 5 minutes using an activity measuring system (Orchid Scientific & Innovative India Pvt. Ltd; Model: ACT01). The displayed values for each rat were documented in the raw data and presented in the report.
Sacrifice and pathology:
BLOOD COLLECTION
At the end of the treatment period, blood samples were collected from all adult animals (i.e. on day 29 of experiment for males and on PND 14 for females). The blood samples were collected after overnight fasting and after anaesthetization with isoflurane. The blood samples were collected from the retro-orbital sinus plexus with a glass capillary tube. Serum thyroid hormone levels were examined as described above.
From randomly selected 5 males and 5 females in each main group, the blood samples were collected for haematology, coagulation parameters and clinical chemistry analysis. For haematology and coagulation parameters, blood samples were collected in tubes containing 10% dipotassium ethylene diamine tetra acetic acid (K2-EDTA) and sodium citrate, respectively. For clinical chemistry, blood samples were collected in a plain tube, allowed to clot, and the serum was collected after centrifugation for analysis.

HEMATOLOGY
The following haematological parameters were analysed for randomly selected 5 males and 5 females in each group towards the end of the experiment period by using a SCIL Vet ABC haematology analyzer, Horiba Medical ABX S.A., USA.
The following were Haematology parameters:
Sr. No. Parameters Units
1 Total Leukocyte Count (WBC) 103 cells/mm3
2 Erythrocyte Count (RBC) 106 cells/mm3
3 Reticulocytes %
4 Hemoglobin (HGB) g/dL
5 Hematocrit (HCT) %
6 Platelet Count 103 cells/mm3
7 Mean corpuscular volume (MCV) µm3
8 Mean Corpuscular Hemoglobin (MCH) pg/cell
9 Mean Corpuscular Hemoglobin Concentration (MCHC) g/dL
10 Differential Leucocytes Count (DLC) %

Estimation of the differential leucocyte count (DLC) was done by a manual method. A blood smear was prepared using standard techniques and stained with Giemsa stain for enumerating the cells. A total of 100 cells were recorded per blood same and the following cell counts were recorded: Lymphocytes (LYM), Monocytes (MON), Neutrophils (NEU), Eosinophils (EOS), and Basophils (BAS). The cell counts were expressed as relative percentages (%). For reticulocyte count, the blood smear was stained using reticulocyte counting reagent. The cell count was expressed in percentage (%). A minimum of 100 cells were counted per blood smear and the following cell count was recorded namely Lymphocytes (LYM), Monocytes (MOM), Neutrophils (NEU), Eosinophils (EOS) and Basophils (BAS).

COAGULATION PARAMETRS
The following coagulation parameters were analysed for randomly selected 5 males and 5 females in each group towards the end of the experiment period by using a Stago Start Max coagulation analyzer (Diagnostica stago, 92600, Asnieres, France).
Blood samples collected for coagulation analysis were centrifuged at 2500 rpm for 10 minutes for separation of plasma. The following were coagulation parameters:
Sr. No. Parameters Unit
1 Prothrombin Time (PT) Seconds
2 Activated Partial Thromboplastin Time (APTT) Seconds
3 Fibrinogen Seconds

CLINICAL CHEMISTRY
The following clinical chemistry parameters were measured for randomly selected 5 males and 5 females in each main group at the end of the experiment period for all surviving animals by using a Randox Daytona Plus, London, UK. Electrolytes were analyzed by using a 9180 Electrolyte Analyzer, Roche, USA. Serum was separated following centrifugation of whole blood samples in a refrigerated centrifuge at 2500 rpm for 10 minutes. Serum samples after the clinical chemistry analysis were stored at -70˚C or colder for thyroid hormone analysis.
The following were clinical chemistry parameters:
Sl. No. Parameters Units
1 Albumin (ALB) g/L
2 Albumin/Globulin ratio (A/G) %
3 Alanine Aminotransferase (ALT) U/L
4 Aspartate Aminotransferase (AST) U/L
5 Alkaline Phosphatase (ALP) U/L
6 Blood Urea Nitrogen (BUN) mg/dL
7 Creatinine (CRE) mg/dL
8 Glucose (Glu) mg/dL
9 Globulin (Glob) mg/dL
10 Serum Urea (URE) mg/dL
11 Total Protein (TP) g/L
12 Total Cholesterol (TCHO) mg/dL
13 Total bile acid µmol/L
Electrolytes
14 Sodium (Na+) mmol/L
15 Potassium (K+) mmol/L

GROSS NECROPSY
All the adult animals in the study were subjected to a full, detailed gross necropsy. Special attention was paid to the organs of the reproductive system. The number of implantation sites were recorded, and vaginal smears were examined on the day of necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.
The testes and epididymides as well as prostate + seminal vesicles with coagulating glands of all adult male animals were collected, trimmed of any adherent tissue, as appropriate, and their wet weight were taken as soon as possible after dissection to avoid drying. Prostate + seminal vesicles with coagulating glands were preserved in 10% neutral buffered formalin (NBF) and testes and epididymides were preserved in the modified Davidson’s fluid. The ovaries and the uterus (with cervix) of all adult female animals were collected, trimmed of any adherent tissue, as appropriate, and their wet weight was taken as soon as possible after dissection to avoid drying and preserved in 10% neutral buffered formalin (NBF) for subsequent histopathological examination.
In addition, from randomly selected 5 adult males and 5 adult females in each group, the liver, kidneys, adrenals, thymus, spleen, brain and heart were trimmed of any adherent tissue, as appropriate and their wet weight was taken as soon as possible after dissection to avoid drying.
The following tissues were preserved in the fixation medium for subsequent histopathological examination: all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, eyes with optic nerve, stomach, small and large intestines (including Peyer's patches), salivary gland, liver, kidneys, adrenals, pancreas, spleen, heart, aorta, thymus, pituitary gland, thyroid with parathyroid, oesophagus, larynx, trachea and lungs (preserved by inflation with fixative and then immersion), gonads (testis and ovaries), accessory sex organs (uterus and cervix, epididymides, prostate, seminal vesicles plus coagulating glands), vagina, urinary bladder, oviducts, rectum, ureters, mesenteric lymph nodes, mandibular lymph node, sciatic nerve in close proximity to the muscle, skeletal muscle, bone and bone marrow smear (femur).
All tissues were preserved in 10% neutral buffered formalin (NBF) (except eyes, testes and epididymides which was preserved in modified Davidson’s fluid).

BSOLUTE AND RELATIVE ORGAN WEIGHTS
Absolute and relative organ weights were recorded for the organs as specified above.

HISTOPATHOLOGY
The detailed histopathological examination was performed on the preserved organs (as specified above) from all adult males and females in G1 and G4. The tissues were processed for routine paraffin embedding and 4 micron sections were stained with Mayer’s hematoxylin and eosin (H&E) staining solution. In addition, testes were sectioned at 4 micron and stained with PAS (Periodic acid–Schiff) reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.
Statistics:
The following statistical methods were used to analyse the body weight, feed consumption, organ weights, functional tests (grip strength, locomotor and landing foot splay), clinical pathology parameters, thyroid hormone levels, as well as reproduction/developmental data.
• Data was summarized in tabular form. Statistical analysis was performed using Graphpad Software Inc, CA, USA (Version: 9.4.1).
• All the data was checked for normality and homogeneity before comparisons.
• All the normally distributed and homogenous data was subjected to One-Way ANOVA followed by Dunnett’s post hoc comparison.
• Data that fails the normality test and/or heterogenous in nature was subjected to Krushkal Wallis ANOVA followed by Dunn’s post hoc comparisons.
• Values were given as mean ± standard deviation (SD).
• All analyses and comparisons were evaluated at the ≤5% (P≤0.05) level.
• All the statistically significant changes were denoted by an asterisk symbol (*) in the summary tables.
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were observed at any dose level.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant changes in body weight or body weight gain were observed at any dose level during the premating and mating period (male/females) or during the gestation and lactation period (females).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No significant changes in food intake were observed at any dose level during the premating and mating period (male/females) or during the gestation and lactation period (females).
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in hematology were observed, except for a significant increase in WBC count in G3 males (mean, 8.94 × 103 cells/ mm3) compared to G1 (mean, 6.80 × 103 cells/ mm3) and a significant decrease in eosinophil count G3 males (mean, 0.60%) compared to G1 (mean, 2.00%). These changes in hematology in G3 males were not considered to be toxicologically relevant due to lack of dose dependency. No significant changes in coagulation parameters were observed in the study.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in clinical chemistry were observed, except for a significant decrease in total cholesterol in G3 males (mean, 50.88 mg/dL) compared to G1 (mean, 69.94 mg/dL), a significant increase in ALT in G2 females (mean, 173.16 U/L) compared to G1 (mean, 124.87 U/L), and a significant increase in ALP in G2 females (mean, 199.33 U/L) compared to G1 (mean, 132.73 U/L). These changes in clinical chemistry in G3 males and in G2 females were not considered to be toxicologically relevant due to lack of dose dependency.
Endocrine findings:
no effects observed
Description (incidence and severity):
For adult male rats, no significant changes in T4 or TSH levels were observed at any dose level.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes in FOB parameters were observed, except for a statistically significant increase in the locomotor activity in terms of Z-movements in the G3 females (mean, 553 counts) compared to G1 females (mean, 484 counts). The above change was considered as incidental and therefore not toxicologically relevant.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In terms of significant changes in absolute and relative organ weight, the follow changes were observed: a significant increase in absolute kidney weight in G2 males (mean, 3.885 gram) compared to G1 (mean, 3.379 gram), a significant increase in absolute heart weight in G4 males (mean, 1.760 gram) compared to G1 males (mean, 1.470 gram), a significant increase in relative heart weight in G3 males (mean, 0.321%) compared to G1 (mean, 0.283%), and a significant increase in relative heart weight in G4 males (mean, 0.324%) compared to G1 (mean, 0.283%). The significant increase in absolute kidney weight in G2 males was considered incidental due to lack of dose dependency. The significant increases in absolute and (or) relative heart weight in G3 and (or) G4 males could not be corroborated with any gross or histopathological findings of the heart in G4 and were therefore not considered to be adverse in nature.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Gross findings were limited to one incidence of unilateral small sized testes and epididymides in one male rat in G3 (Animal No. 050). This finding was considered to be spontaneous in nature and not toxicologically relevant due to lack of dose dependency.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic findings were limited to one incidence of aspermia (unilateral) in the epididymides and atrophy of the seminiferous tubules (unilateral, diffuse, severe), with complete absence of stages of spermatogenesis in the PAS-stained testes slides of the affected area, in one male rat in G3 (Animal No. 050). Tubular degeneration/atrophy in testes and aspermia in epididymides at a low incidence can be considered as a background finding in male rats (see Toxicologic Pathology, 40: 40S-121S, 2012 and “Background lesions in laboratory animals: a Color atlas” edited by EF McInness, Saunders Elsevier, Edinburgh, 2011, page 102, ISBN: 978-0-7020-3519-7). In due to this, and considering lack of dose dependency, the observed microscopic findings in the male rat in G3 (Animal No. 050) was considered to be incidental in nature and not toxicologically relevant. The stage of estrous cycle of all the adult females in G1 and G4 groups at the terminal sacrifice were in correlation with the histopathology of respective female reproductive organs.
Histopathological findings: neoplastic:
no effects observed
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:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
serum/plasma biochemistry
serum/plasma hormone analyses
Key result
Critical effects observed:
no

Table 1 - SUMMARY OF MORTALITY/MORBIDITY





















































































GroupsDose(mg/kg bw/day)SexPre-terminal deaths
Deaths during pre-mating and mating periodDeaths during gestationDeaths during lactationMoribund sacrificeTotal deaths
G10M0NANA00
F00000
G2100M0NANA00
F00000
G3300M0NANA00
F00000
G41000M0NANA00
F00000

 


Table 2 - SUMMARY OF CLINICAL SIGNS – MALES



































GroupsDose(mg/kg bw/day)SexNo. of animals/Total no. treated animalsClinical Signs
G10M10/10Normal
G2100M10/10Normal
G3300M10/10Normal
G41000M10/10Normal

 


Table 3 - SUMMARY OF CLINICAL SIGNS – FEMALES



































GroupsDose(mg/kg bw/day)SexNo. of animals/Total no. treated animalsClinical Signs
G10F10/10Normal
G2100F10/10Normal
G3300F10/10Normal
G41000F10/10Normal

 


Table 4- SUMMARY OF DETAILED CLINICAL EXAMINATION – MALES









































































































































































































Home cage observationDaysGroups / Dose (mg/kg bw/day)
G1 / 0G2 / 100G3 / 300G4 / 1000
 Animal body position/Posture
R - RearingS - Sitting or Standing normallyA - Asleep		Prior to Treatment3R,5S,2A4R,5S,1A3R,5S,2A3R,5S,2A
83R,7S,0A4R,5S,1A3R,6S,1A2R,7S,1A
153R,5S,2A3R,5S,2A3R,6S,1A3R,6S,1A
223R,6S,1A4R,5S,1A2R,6S,2A4R,5S,1A
283R,5S,2A3R,6S,1A3R,5S,2A4R,5S,1A
Respiration - NormalPrior to Treatment, 8, 15, 22, 28 10/1010/1010/1010/10
Vocalization - Normal10/1010/1010/1010/10
Hand-held observation
Ease of removal - Easy/NormalPrior to Treatment, 8, 15, 22, 2810/1010/1010/1010/10
Handling reactivity- Easy to handle/ normal10/1010/1010/1010/10
Palpebral closure - Open10/1010/1010/1010/10
Lacrimation - No lacrimation10/1010/1010/1010/10
Nasal discharge - No discharge10/1010/1010/1010/10
Salivation - No salivation10/1010/1010/1010/10
Teeth - Normal10/1010/1010/1010/10
Fur coat/ Skin - Normal10/1010/1010/1010/10
Muscle tone/ Mass - Normal10/1010/1010/1010/10
Perineum wetness - None/normal10/1010/1010/1010/10
Tail - Normal10/1010/1010/1010/10
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walkingPrior to Treatment, 8, 15, 22, 2810/1010/1010/1010/10
Arousal - Normal10/1010/1010/1010/10
Clonic convulsion - None/ normal10/1010/1010/1010/10
Tonic Convulsion - None/ normal10/1010/1010/1010/10
Stereotype - None10/1010/1010/1010/10
Bizarre /Abnormal behaviour(s) - None10/1010/1010/1010/10
Fecal consistency - Formed/ normal10/1010/1010/1010/10

 


TABLE 4 - (CONTD.) - SUMMARY OF DETAILED CLINICAL EXAMINATION – MALES                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            

















































































































































































































































Rearing count
Group / Dose (mg/kg bw/day) Prior to Treatment8152228
G1 / 0Mean17.7011.7013.2012.1011.90
SD2.411.732.821.371.14
G2 / 100Mean17.7012.1011.9011.8012.40
SD1.420.702.740.751.02
G3 / 300Mean17.9011.3014.5011.8012.40
SD1.921.423.641.171.11
G4 / 1000Mean18.0011.7011.6011.8012.10
SD1.551.792.010.981.30
Urine pools (count)
G1 / 0 Mean3.904.003.003.003.10
SD1.220.631.260.770.83
G2 / 100Mean3.703.702.703.702.70
SD1.190.900.900.900.90
G3 / 300Mean3.703.502.103.802.70
SD1.421.360.830.870.78
G4 / 1000Mean3.403.102.903.002.90
SD0.920.700.700.770.83
Fecal (count) 
G1 / 0 Mean1.401.801.201.702.00
SD0.920.870.400.640.63
G2 / 100Mean2.201.801.401.901.70
SD1.250.600.490.700.64
G3 / 300Mean1.401.701.301.801.40
SD0.920.640.460.750.49
G4 / 1000Mean1.601.901.301.801.30
SD1.020.830.460.750.46
            

 


Table 5 - SUMMARY OF DETAILED CLINICAL EXAMINATION – FEMALES DURING PRE MATING PERIOD



























































































































































































Home cage observationDaysGroups / Dose (mg/kg bw/day)
G1 / 0G2 / 100G3 / 300G4 / 1000
 Animal body position/Posture
R - RearingS - Sitting or Standing normallyA - Asleep		Prior to Treatment4R,5S,1A2R,6S,2A4R,4S,2A3R,6S,1A
82R,6S,2A4R,5S,1A3R,6S,1A3R,6S,1A
153R,6S,1A3R,7S,0A3R,6S,1A3R,7S,0A
Respiration - NormalPrior to Treatment, 8, 15,   10/1010/1010/1010/10
Vocalization - Normal10/1010/1010/1010/10
Hand-held observation
Ease of removal - Easy/NormalPrior to Treatment, 8, 15,   10/1010/1010/1010/10
Handling reactivity- Easy to handle/ normal10/1010/1010/1010/10
Palpebral closure - Open10/1010/1010/1010/10
Lacrimation - No lacrimation10/1010/1010/1010/10
Nasal discharge - No discharge10/1010/1010/1010/10
Salivation - No salivation10/1010/1010/1010/10
Teeth - Normal10/1010/1010/1010/10
Fur coat/ Skin - Normal10/1010/1010/1010/10
Muscle tone/ Mass - Normal10/1010/1010/1010/10
Perineum wetness - None/normal10/1010/1010/1010/10
Tail - Normal10/1010/1010/1010/10
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walkingPrior to Treatment, 8, 15,   10/1010/1010/1010/10
Arousal - Normal10/1010/1010/1010/10
Clonic convulsion - None/ normal10/1010/1010/1010/10
Tonic Convulsion - None/ normal10/1010/1010/1010/10
Stereotype - None10/1010/1010/1010/10
Bizarre /Abnormal behaviour(s) - None10/1010/1010/1010/10
Fecal consistency - Formed/ normal10/1010/1010/1010/10

 


TABLE 5 – (CONTD.) -   DETAILED CLINICAL EXAMINATION – FEMALES DURING PRE MATING PERIOD                                                                                        
















































































































































































Rearing count
Group / Dose          (mg/kg bw/day)14815
G1 / 0Mean17.8013.1011.70
SD1.942.622.15
G2 / 100Mean18.1011.2012.20
SD1.582.041.99
G3 / 300Mean17.3011.4011.50
SD2.102.331.80
G4 / 1000Mean17.4010.6012.40
SD1.501.852.80
Urine pools (count)
G1 / 0Mean3.404.002.00
SD1.360.630.77
G2 / 100Mean3.503.802.60
SD1.280.750.80
G3 / 300Mean3.003.002.10
SD1.000.771.04
G4 / 1000Mean4.302.902.20
SD1.191.220.98
Fecal (count)
G1 / 0Mean1.702.101.30
SD1.190.700.46
G2 / 100Mean1.601.801.30
SD1.020.870.46
G3 / 300Mean1.501.801.20
SD0.920.750.40
G4 / 1000Mean1.501.601.30
SD0.810.800.46

 


TABLE 5 (CONTD.) SUMMARY OF DETAILED CLINICAL EXAMINATION DURING GESTATION PERIOD                                 


































































































































































































Home cage observationDaysGroups / Dose (mg/kg bw/day)
G1 / 0G2 / 100G3 / 300G4 / 1000
 Animal body position/Posture R - RearingS - Sitting or Standing normallyA - Asleep03R,7S.0A4R,6S,0A2R,8S,0A3R,7S.0A
74R,6S,0A4R,6S,0A5R,5S,0A5R,5S,0A
144R,6S,0A4R,6S,0A5R,5S,0A2R,8S,0A
205R,5S,0A3R,7S.0A5R,5S,0A3R,7S.0A
Respiration - Normal0,7,14,2010/1010/1010/1010/10
Vocalization - Normal10/1010/1010/1010/10
Hand-held observation
Ease of removal - Easy/Normal0,7,14,2010/1010/1010/1010/10
Handling reactivity- Easy to handle/ normal10/1010/1010/1010/10
Palpebral closure - Open10/1010/1010/1010/10
Lacrimation - No lacrimation10/1010/1010/1010/10
Nasal discharge - No discharge10/1010/1010/1010/10
Salivation - No salivation10/1010/1010/1010/10
Teeth - Normal10/1010/1010/1010/10
Fur coat/ Skin - Normal10/1010/1010/1010/10
Muscle tone/ Mass - Normal10/1010/1010/1010/10
Perineum wetness - None/normal10/1010/1010/1010/10
Tail - Normal10/1010/1010/1010/10
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking0,7,14,2010/1010/1010/1010/10
Arousal - Normal10/1010/1010/1010/10
Clonic convulsion - None/ normal10/1010/1010/1010/10
Tonic Convulsion - None/ normal10/1010/1010/1010/10
Stereotype - None10/1010/1010/1010/10
Bizarre /Abnormal behaviour(s) - None10/1010/1010/1010/10
Fecal consistency - Formed/ normal10/1010/1010/1010/10

 


TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL EXAMINATION DRUNG GESTATION PERIOD












































































































































































































Open- field observation (contd.)
Rearing count
Group / Dose       (mg/kg bw/day)071420
G1 / 0 Mean9.209.009.309.20
SD1.991.951.952.48
G2 / 100Mean9.009.209.209.40
SD1.262.091.762.76
G3 / 300Mean10.8011.009.9010.00
SD2.232.491.041.73
G4 / 1000Mean8.909.107.807.80
SD2.171.641.081.72
Urine pools (count)
G1 / 0 Mean1.701.701.901.50
SD0.640.780.540.81
G2 / 100Mean2.001.902.002.00
SD0.770.700.890.63
G3 / 300Mean1.501.801.801.80
SD0.810.600.600.75
G4 / 1000Mean1.901.802.102.10
SD0.700.750.700.94
Fecal (count)
G1 / 0 Mean1.301.001.101.10
SD0.900.890.700.94
G2 / 100Mean1.001.101.101.00
SD0.770.700.830.77
G3 / 300Mean1.001.101.001.20
SD0.630.700.770.60
G4 / 1000Mean1.001.001.001.30
SD0.630.770.771.19

              


TABLE 5 (CONTD.) SUMMARY OF DETAILED CLINICAL EXAMINATION DURING LACTATION PERIOD




















































































































































































Home cage observationDaysGroups / Dose (mg/kg bw/day)
G1 / 0G2 / 100G3 / 300G4 / 1000
63R,7S,0A6R,4S,0A3R,7S,0A4R,6S,0A
Animal body position/PostureR - RearingS - Sitting or Standing normallyA - Asleep133R,7S,0A4R,6S,0A4R,4S,0A6R,4S,0A
Respiration - Normal6,1310/1010/1010/1010/10
Vocalization - Normal10/1010/1010/1010/10
Hand-held observation
Ease of removal - Easy/Normal6,1310/1010/1010/1010/10
Handling reactivity- Easy to handle/ normal10/1010/1010/1010/10
Palpebral closure - Open10/1010/1010/1010/10
Lacrimation - No lacrimation10/1010/1010/1010/10
Nasal discharge - No discharge10/1010/1010/1010/10
Salivation - No salivation10/1010/1010/1010/10
Teeth - Normal10/1010/1010/1010/10
Fur coat/ Skin - Normal10/1010/1010/1010/10
Muscle tone/ Mass - Normal10/1010/1010/1010/10
Perineum wetness - None/normal10/1010/1010/1010/10
Tail - Normal10/1010/1010/1010/10
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking6,1310/1010/1010/1010/10
Arousal - Normal10/1010/1010/1010/10
Clonic convulsion - None/ normal10/1010/1010/1010/10
Tonic Convulsion - None/ normal10/1010/1010/1010/10
Stereotype - None10/1010/1010/1010/10
Bizarre /Abnormal behaviour(s) - None10/1010/1010/1010/10
Fecal consistency - Formed/ normal10/1010/1010/1010/10

           


TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL EXAMINATION DRUNG LACTATION PERIOD                                                     


























































































































































Open- field observation (contd.)
Rearing count
Group / Dose          (mg/kg bw/day)613
G1 / 0 Mean9.0010.10
SD2.282.88
G2 / 100Mean9.4010.50
SD2.372.33
G3 / 300Mean9.6010.20
SD2.202.40
G4 / 1000Mean9.309.40
SD2.451.80
Urine pools (count)
G1 / 0 Mean2.702.60
SD0.780.66
G2 / 100Mean2.202.00
SD0.750.45
G3 / 300Mean2.202.40
SD0.750.49
G4 / 1000Mean2.702.10
SD0.640.54
Fecal (count)
G1 / 0 Mean1.301.60
SD0.640.49
G2 / 100Mean1.801.40
SD0.400.80
G3 / 300Mean1.701.50
SD0.640.50
G4 / 1000Mean1.601.60
SD0.490.80

 


Table 6 - SUMMARY OF BODY WEIGHTS – MALES                                                                                                     























































































































Average Body Weights (g) - Males
Days
GroupsDose(mg/kg bw/day)18152228
G10Mean494.56511.72525.22530.52537.32
SD19.9825.4528.1029.0530.00
N1010101010
G2100Mean495.04517.29533.39541.02546.86
SD17.8222.7625.1124.5625.39
N1010101010
G3300Mean493.32510.23525.12530.30536.93
SD19.2523.0328.5323.9127.29
N1010101010
G41000Mean492.97516.92534.46538.12542.35
SD20.0024.6827.1829.0529.35
N1010101010

       Key: Values are in Mean ± SD.


   Table 7 - SUMMARY OF NET BODY WEIGHT GAIN (g) – MALES                                                                                                                                                                                                          































































































































GroupsDose(mg/kg bw/day)FromTo1881515222228Abs Gain128TotalGain %128
G10Mean17.1613.505.306.8042.768.62
SD9.795.939.616.5816.723.22
N101010101010
G2100Mean22.2516.117.635.8351.8210.45
SD9.616.499.854.4112.642.41
N101010101010
G3300Mean16.9114.895.186.6343.618.82
SD5.987.239.696.5914.202.75
N101010101010
G41000Mean23.9517.543.664.2449.3810.00
SD6.285.2710.946.1717.423.44
N101010101010

        Key: Values are in Mean ± SD; g =gram


TABLE 8 - SUMMARY OF BODY WEIGHTS DURING PRE MATING PERIOD – FEMALES





























































































Body Weights (g) During Pre Mating Period – Females
Days
GroupsDose(mg/kg bw/day)1815
G10Mean272.96278.01280.24
SD16.3817.2417.42
N101010
G2100Mean271.97278.32280.72
SD15.4415.8916.80
N101010
G3300Mean270.46277.83281.79
SD17.3415.1614.70
N101010
G41000Mean272.86278.23282.42
SD15.5315.0212.66
N101010

Key: Values are in Mean ± SD ; g =gram.


 


TABLE 9 - SUMMARY OF NET BODY WEIGHT GAIN (g) DURING PRE MATING PERIOD – FEMALES





































































































GroupsDose(mg/kg bw/day)FromTo18815Abs. Gain115Total% Gain115
G10Mean7.282.685.062.23
SD7.132.655.225.71
N10101010
G2100Mean8.763.226.352.40
SD4.401.614.092.26
N10101010
G3300Mean11.334.297.383.96
SD7.312.868.034.83
N10101010
G41000Mean9.563.605.364.20
SD6.962.827.853.58
N10101010

Key: Values are in Mean ± SD; g =gram


 


TABLE 10 - SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE – MALES (PRE MATING PERIOD)













































































GroupsDose(mg/kg bw/day)Average Cage Wise Food Intake (g/rat/day)
12
G10Mean29.1228.64
SD1.872.12
N1010
G2100Mean29.5729.31
SD0.411.28
N1010
G3300Mean28.6027.95
SD2.051.68
N1010
G41000Mean29.8129.72
SD1.360.77
N1010

 Key: Values are in Mean ± SD; g =gram


 


TABLE 11 - SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE (PRE MATING PERIOD) – FEMALES                                                                                                                                                                  













































































GroupsDose(mg/kg bw/day)Weeks (g/rat/day)
                        12
G10Mean19.2719.55
SD0.830.76
N1010
G2100Mean19.0518.51
SD1.651.58
N1010
G3300Mean18.5219.12
SD0.721.19
N1010
G41000Mean18.7319.55
SD1.341.38
N1010

 Key: Values are in Mean ± SD ; g =gram.


 


TABLE 12 - SUMMARY OF MATERNAL BODY WEIGHTS (g) AND BODY WEIGHT CHANGES DURING GESTATION PERIOD




























































































































































GroupsDose(mg/kg bw/day)Body weight on Gestation (Day) Body weight change during Gestation (Day)
0714200-77-1414-200-20
G10Mean283.99309.75345.55432.9825.7635.8087.43148.99
SD16.4514.3319.1721.398.1710.1217.1719.70
N1010101010101010
G2100Mean285.41312.77349.29429.7127.3636.5280.42144.31
SD18.2223.4420.8134.0517.3214.9925.0630.59
N1010101010101010
G3300Mean287.93317.61350.14443.3029.6832.5393.16155.37
SD14.0716.6317.9821.7915.035.2010.3518.99
N1010101010101010
G41000Mean287.75311.80351.17442.5924.0439.3891.42154.84
SD15.7015.7616.8723.545.7111.0814.0116.53
N1010101010101010

 Key: Values are in Mean ± SD ; g =gram


 


Table 13 - SUMMARY OF MATERNAL FOOD INTAKE DURING GESTATION PERIOD                                                                                                       











































































































Maternal food intake (g/rat/day) during Gestation period
Gestation Period (Day)
GroupsDose(mg/kg bw/day)FromTo077141420020
G10Mean25.3227.5431.1328.00
SD1.283.632.971.21
N10101010
G2100Mean25.1327.5829.5027.40
SD2.022.602.290.29
N10101010
G3300Mean24.4627.4329.9227.27
SD1.043.332.121.15
N10101010
G41000Mean24.8527.6131.5528.00
SD1.732.211.810.26
N10101010

 Key: Values are in Mean ± SD ; g =gram


 


Table 14 -SUMMARY OF MATERNAL BODY WEIGHTS DURING THE LACTATION PERIOD





















































































































GroupsDose(mg/kg bw/day)Body weight (g) during Lactation period (Day)Body weight change during Lactation period (Day)
04130 - 44 – 13
G10Mean326.11328.96342.982.8614.01
SD21.8020.5021.3610.908.54
N1010101010
G2100Mean326.99331.00346.824.0115.83
SD30.7027.8229.0812.519.61
N1010101010
G3300Mean335.79337.75347.581.969.83
SD23.8827.3624.558.1515.21
N1010101010
G41000Mean332.16334.35340.022.195.67
SD13.5517.9518.2711.5911.72
N1010101010

 Key: Values are in Mean ± SD; g =gram


 


Table 15 - SUMMARY OF MATERNAL FOOD INTAKE DURING THE LACTATION PERIOD
















































































GroupsDose(mg/kg bw/day)Maternal food intake (g/rat/day) during Lactation period
Per day during Lactation (Day)
                                  0 - 44 - 13
G10Mean37.7338.04
SD4.760.75
N1010
G2100Mean35.8438.06
SD1.070.38
N1010
G3300Mean35.5337.89
SD1.510.49
N1010
G41000Mean36.6137.79
SD2.650.93
N1010

 Key: Values are in Mean ± SD; g =gram


Table 16 - SUMMARY OF THYROID HORMONE ANALYSIS – MALES/SIRES                                                                      


















































































                                                                                                                                                                Parameters
GroupsDose(mg/kg bw/day) T4TSH
ng/mLpg/mL
G10Mean43.97517.28
SD3.03121.44
N1010
G2100Mean41.20571.26
SD5.28109.88
N1010
G3300Mean41.33529.77
SD2.0660.14
N1010
G41000Mean43.49435.47
SD3.9768.28
N1010

 Key: Values are in Mean ± SD


 


Table 17 - SUMMARY OF OESTOUS CYCLICITY – PRE MATING











































GroupsDose(mg/kg. bw/day)Mean cycle length (days)No. of animals with normal cycleNo. of animals with abnormal cycle 
 
G1 04.05100 
G21004.10100 
G33004.03100 
G410004.00100 

 


TABLE 18 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)



















































































ParameterGroups (N) 
G1G2G3G4
Home cage observations
Posture
Sitting or standing normally4223
Standing on hind limbs1332
Asleep (lying on side)----
Respiration
Normal5555
Clonic involuntary movement
None/normal5555
Tonic involuntary movement
None/normal5555
      

 


TABLE 18 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)














































































ParameterGroups (N)
G1G2G3G4
Vocalization
None/normal5555
Convulsions
Absent – N5555
Hand-held observations
Reactivity
Easy/ normal (animal does not resist)5555
Handling
Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal 5555
Palpebral closure
Open5555
Lacrimation
No lacrimation/ normal5555

 


TABLE 18 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES) 











































































ParameterGroups (N)
G1G2G3G4
Salivation
No salivation/ normal5555
Piloerection
None/ normal5555
Perineum Wetness
N: Absence of wetness5555
Body Temperature Range (ºC)36.34  ± 0.2336.50 ± 0.5436.24 ± 0.5436.32 ± 0.46
Open Field Activity
Clonic involuntary movement
None/ Normal5555
Tonic involuntary movement
None/ Normal5555

 


TABLE 18 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)


























































































































ParameterGroups (N) 
G1G2G3G4
Gait
N:Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking5555
Movements
Normal5555
Arousal
Normal (keeps guard up and engages in exploratory activity)5555
Tremors
None5555
Occurrence of stereotype0000
Abnormal behaviour0000
Number of Defecations (Mean)1.20 ± 0.841.00 ± 0.711.40 ± 0.551.00 ± 0.71
Number of Urinations (Mean)2.40 ± 0.551.80 ± 0.841.80 ± 0.842.40 ± 0.89
Number of Rearings (Mean)8.80 ± 2.179.00 ± 2.358.40 ± 2.078.40 ± 1.67
Sensory Reactivity Measurements
Approach Response
Slowly approaches, sniffs and pulls back/ normal5555
Touch Response
Slowly approaches, sniffs and pulls back/ normal5555
      

 


TABLE 18 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)








































































































ParameterGroups (N)
G1G2G3G4
Eyelid reflex
Blink/ Normal5555
Pinna reflex
Auricle twitches/ normal5555
Sound response
Mild reaction, hears sound/normal5555
Tail pinch response
Looks back, moves forward and lightly squeaks/normal5555
Pupillary reflex
Contracts5555
Aerial righting reflex
Lands on four limbs/ normal5555
Tail limb reflex
Present (Y)5555
Abdominal tone
Normal (proper hardness)5555
       

 


TABLE 18 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)






















































































ParameterGroups (n) 
G1G2G3G4
Limb tone
Normal (proper hardness)5555
Landing Foot Splay (Mean ± SD (mm)54.93 ± 2.8264.93 ± 11.0762.53 ± 8.5258.67 ± 11.02
Grip strength fore limb (Mean ± SD (g/f)705.2 ± 25.6701.3 ± 41.7723.8 ± 49.9709.2 ± 48.8
Grip strength hind limb (Mean ± SD (g/f)438.0 ± 13.8432.9 ± 14.5 446.3 ± 18.5 448.8 ± 27.5
Locomotor activity (Mean ± SD)
X - Movements521 ± 50493 ± 61507 ± 34529 ± 36
Y - Movements502 ± 19484 ± 91491 ± 54498 ± 69
Z - Movements452 ± 34488 ± 19453 ± 51426 ± 86
Total Movements (X+Y+Z)1475 ± 641465 ± 1271451 ± 631453 ± 150
      

 Key: Values are in Mean ± SD.


 


TABLE 19 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)










































































ParameterGroups (N)
G1G2G3G4
Home cage observations
Posture
Sitting or standing normally3332
Standing on hind limbs2223
Asleep (lying on side)0000
Respiration
Normal5554
Clonic involuntary movement
None/normal5554
Tonic involuntary movement
None/normal5554

 


TABLE 19 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)














































































ParameterGroups (N)
G1G2G3G4
Vocalization
None/normal5555
Convulsions
Absent – N5555
Hand-held observations
Reactivity
Easy/ normal (animal does not resist)5555
Handling
Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal 5555
Palpebral closure
Open5555
Lacrimation
No lacrimation/ normal5555

 


TABLE 19 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES) 











































































ParameterGroups (N)
G1G2G3G4
Salivation
No salivation/ normal5555
Piloerection
None/ normal5555
Perineum Wetness
N: Absence of wetness5555
Body Temperature Range (ºC)36.96 ± 0.4536.98 ± 0.5036.92 ± 0.5136.88 ± 0.52
Open Field Activity
Clonic involuntary movement
None/ Normal5555
Tonic involuntary movement
None/ Normal5555

 


TABLE 19 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

















































































































ParameterGroups (N)
G1G2G3G4
Gait
N:Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking5555
Movements
Normal5555
Arousal
Normal (keeps guard up and engages in exploratory activity)5555
Tremors
None5555
Occurrence of stereotype0000
Abnormal behaviour0000
Number of Defecations (Mean)1.60 ± 1.522.00 ± 1.222.00 ± 0.712.20 ± 0.84
Number of Urinations (Mean)3.60 ± 1.142.00 ± 1.223.00 ± 0.712.60 ± 1.14
Number of Rearings (Mean)20.00± 2.0019.40 ± 2.1918.80 ± 3.1920.00 ± 2.24
Sensory Reactivity Measurements
Approach Response
Slowly approaches, sniffs and pulls back/ normal5554
Touch Response
Slowly approaches, sniffs and pulls back/ normal5554

 


TABLE 19 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)































































































ParameterGroups (N)
G1G2G3G4
Eyelid reflex
Blink/ Normal5555
Pinna reflex
Auricle twitches/ normal5555
Sound response
Mild reaction, hears sound/normal5555
Tail pinch response
Looks back, moves forward and lightly squeaks/normal5555
Pupillary reflex
Contracts5555
Aerial righting reflex
Lands on four limbs/ normal5555
Tail limb reflex
Present (Y)5555
Abdominal tone
Normal (proper hardness)5555

 


TABLE 19 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)













































































ParameterGroups (N)
G1G2G3G4
Limb tone
Normal (proper hardness)5555
Landing Foot Splay(Mean ± SD (mm)64.33 ± 14.5060.80 ± 13.7967.13 ± 3.0275.27 ± 21.55
Grip strength fore limb(Mean ± SD (g/f)677.5 ± 16.4664.6 ± 22.5681.4 ± 3.7697.6 ± 91.1
Grip strength hind limb(Mean ± SD (g/f)404.5 ± 21.7 422.5 ± 14.3429.1 ± 13.8418.7 ± 13.9
Locomotor activity (Mean ± SD)
X - Movements459 ± 61493 ± 12456 ± 40520 ± 54
Y - Movements555 ± 43550 ± 28543 ± 37548 ± 63
Z - Movements 484 ± 70541 ± 33553 ± 25* 526 ± 83
Total Movements (X+Y+Z)1506 ± 1691584 ± 211553 ± 571594 ± 179

 Key: Values are in Mean ± SD; * - Statistically significant from vehicle control (p ≤ 0.05)


 


Table 20 – SUMMARY OF HAEMATOLOGICAL PARAMETERS - MALES 

























































































































































































































































GroupDose(mg/kg bw/day) Haematological Parameters
WBCRBCHbHCTPLTMCVMCHMCHCNEULYMEOSMONBASRetic
103 cells/ mm3106 cells/ mm3g/dL%103 cells/ mm3µm3pg/cellg/dL%%%%%%
G10Mean6.808.9615.7845.92865.8051.0017.6434.3825.6072.002.000.400.002.54
SD0.980.400.511.1659.651.580.770.544.344.470.000.890.000.38
N55555555555555
G2100 Mean8.628.6016.0045.40915.6053.0018.6235.2025.0073.800.800.400.002.58
SD1.390.350.391.30125.491.580.860.826.786.870.450.550.000.18
N55555555555555
G3300Mean8.94*8.8115.8445.52860.4051.8018.0234.8424.4074.600.60*0.400.002.46
SD1.250.370.231.1371.901.640.530.634.104.980.550.550.000.15
N55555555555555
G41000Mean7.368.8115.6445.14992.0051.2017.7634.6824.0075.001.000.000.002.58
SD1.530.540.772.79116.770.840.310.585.105.001.000.000.000.29
N55555555555555

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05).


 


Table 21 – SUMMARY OF HAEMATOLOGICAL PARAMETERS - FEMALES

























































































































































































































































GroupDose(mg/kg bw/day) Haematological Parameters
WBCRBCHbHCTPLTMCVMCHMCHCNEULYMEOSMONBASRetic
103 cells/ mm3106 cells/mm3g/dL%103 cells/ mm3µm3Pg/cellg/dL%%%%%%
G10Mean5.587.2214.7042.34992.8058.8020.3834.7429.4069.001.000.600.002.54
SD1.480.460.853.18197.232.050.440.718.057.871.000.890.000.27
N55555555555555
G2100 Mean6.607.4714.7443.201038.8058.0019.7834.1629.2069.600.800.400.002.76
SD1.920.410.511.13109.702.350.920.641.100.890.840.550.000.15
N55555555555555
G3300Mean5.547.2914.2841.48944.0057.0019.6234.4429.2068.601.600.800.002.90
SD1.070.610.683.00176.360.710.831.222.282.610.550.840.000.34
N55555555555555
G41000Mean6.067.6515.2644.88913.4058.6020.0034.2030.2067.601.400.800.002.36
SD3.381.713.0610.67290.241.950.431.472.864.341.140.840.000.38
N55555555555555

Key: Values are in Mean ± SD


 


TABLE 22 - SUMMARY OF COAGULATION PARAMTERS - MALES































































































GroupsDose(mg/kg bw/day)Coagulation Parameters
PTAPTTFN
SecondsSecondsmg/dL
G10Mean13.9020.58260.32
SD2.141.3541.22
N555
G2100 Mean13.3821.40242.56
SD2.022.0546.03
N555
G3300Mean14.9024.22277.60
SD1.654.0423.17
N555
G41000Mean14.9023.66249.45
SD3.044.1530.15
N555

Key: Values are in Mean ± SD.


 


TABLE 23 - SUMMARY OF COAGULATION PARAMTERS - FEMALES































































































GroupsDose(mg/kg bw/day)Coagulation Parameters
PTAPTTFN
SecondsSecondsmg/dL
G10Mean12.4219.02234.84
SD1.783.3729.22
N555
G2100Mean13.5621.42256.55
SD3.556.8842.89
N555
G3300Mean13.1820.14255.95
SD2.093.9225.75
N555
G41000Mean11.3617.96248.73
SD0.852.5317.35
N555

Key: Values are in Mean ± SD.


 


TABLE 24- SUMMARY OF CLINICAL CHEMISTRY PARAMETERS – MALES

































































































































































































GroupsDose(mg/kg bw/day) Clinical Chemistry Parameters
GLUUREACRETCHOASTALTTPALBGLOBTBA
mg/dLmg/dLmg/dLmg/dLU/LU/Lg/Lg/Lg/Lµmol/L
G10Mean102.1634.650.7669.94138.4464.1264.6941.7822.9131.80
SD14.194.190.0711.8917.148.883.881.882.205.24
N5555555555
G2100 Mean112.6633.530.8252.98159.3778.9160.7839.5721.2131.57
SD15.803.150.0711.4729.2926.572.551.401.939.84
N5555555555
G3300Mean108.6935.760.7450.88*141.8669.7962.7040.5022.2026.80
SD5.422.050.047.7013.529.022.070.832.028.77
N5555555555
G41000Mean109.0930.280.7457.72133.8767.5063.6741.0722.6037.83
SD16.114.150.0213.5012.4716.163.271.471.959.62
N5555555555

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05).


 


TABLE 24 (CONTD.) - SUMMARY OF CLINICAL CHEMISTRY PARAMETERS – MALES



























































































































GroupsDose(mg/kg bw/day)Clinical Chemistry Parameters
ALPNa+K+BUNA/G ratio
U/Lmmol/Lmmol/Lmg/dL-
G10Mean69.55141.604.5616.191.83
SD12.120.550.431.960.13
N55555
G2100 Mean76.00140.804.9415.671.88
SD5.520.840.361.470.17
N55555
G3300Mean86.84140.804.3216.711.84
SD17.321.790.190.960.18
N55555
G41000Mean86.94142.004.6214.151.82
SD11.031.000.901.940.12
N55555

Key: Values are in Mean ± SD


 


   TABLE 25 - SUMMARY OF CLINICAL CHEMISTRY PARAMETERS – FEMALES

































































































































































































GroupsDose(mg/kg bw/day) Clinical Chemistry Parameters
GLUUREACRETCHOASTALTTPALBGLOBTBA
mg/dLmg/dLmg/dLmg/dLU/LU/Lg/Lg/Lg/Lµmol/L
G10Mean70.2057.280.59126.09207.34124.8758.2023.8634.35116.31
SD10.2810.840.0520.4361.2026.198.334.633.7241.07
N5555555555
G2100 Mean78.6650.410.55125.94261.82173.16*58.5326.3232.2176.20
SD24.4014.670.0630.7775.4523.0810.351.5310.0547.07
N5555555555
G3300Mean73.5449.280.59113.39206.80130.5161.6625.5636.1093.80
SD8.3415.120.0434.4634.1721.265.832.923.7677.20
N5555555555
G41000Mean90.2651.950.60114.84169.94106.6754.7326.3928.3363.18
SD18.2612.120.0813.1667.8223.5811.461.9310.7340.71
N5555555555

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05).


 


TABLE 25 (CONTD.) - SUMMARY OF CLINICAL CHEMISTRY PARAMETERS – FEMALES



























































































































GroupsDose(mg/kg bw/day)Clinical Chemistry Parameters
ALPNa+K+BUNA/G ratio
U/Lmmol/Lmmol/Lmg/dL-
G10Mean132.73142.205.4426.770.69
SD20.693.490.185.070.06
N55555
G2100 Mean199.33*139.804.8023.560.93
SD40.101.480.646.850.47
N55555
G3300Mean106.03139.205.4823.030.71
SD24.802.170.377.070.08
N55555
G41000Mean131.31139.805.0424.281.07
SD33.013.110.385.670.49
N55555

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05).


 


TABLE 26 - SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS– MALES





































































































































































GroupsDose(mg/kg bw/day)Fasting Body Weight (FBW) and Absolute Organ Weights
FBWLiverKidneysAdrenalsSpleenThymusHeartBrain
(g)(g)(g)(g)(g)(g)(g)(g)
G10Mean520.0215.5663.3790.1080.9990.6531.4702.159
SD41.520.6270.3060.0210.1620.0770.1130.108
N55555555
G2100 Mean546.3116.3523.885*0.1081.0800.6111.6382.256
SD27.340.6810.2550.0070.1840.0950.1540.151
N55555555
G3300Mean528.5316.3923.4800.1001.0740.6791.6942.241
SD27.540.6210.2950.0060.1370.0740.1350.141
N55555555
G41000Mean544.1316.4343.8300.1151.0020.6051.760*2.191
SD32.500.9460.2420.0270.0930.0680.1100.100
N55555555

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05).


 


 


TABLE 26 (CONTD.)- SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS– MALES



























































































































GroupsDose(mg/kg bw/day)Fasting Body Weight (FBW) and Absolute Organ Weights (g)
FBWTestesEpididymisProstate+ Seminal vesicles with Coagulating glandsThyroid with Parathyroid
(g)(g)(g)(g)(g)
G10Mean523.234.1581.7493.3560.357
SD30.240.2110.1130.3640.041
N1010101010
G2100 Mean534.204.1471.6523.4510.346
SD25.190.3630.1540.3340.041
N1010101010
G3300Mean525.114.1241.6653.4340.325
SD25.690.2090.2120.2460.060
N1010101010
G41000Mean532.604.1731.7433.5080.351
SD28.830.4380.1750.4110.061
N1010101010

Key: Values are in Mean ± SD.


 


TABLE 27 - SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS – FEMALES





































































































































































GroupsDose(mg/kg bw/day)Fasting Body Weight (FBW) and Absolute Organ Weights
FBWLiverKidneysAdrenalsSpleenThymusHeartBrain
(g)(g)(g)(g)(g)(g)(g)(g)
G10Mean326.9416.5732.3350.1000.9290.3301.1912.034
SD13.891.2000.2010.0060.1960.0230.1180.034
N55555555
G2100 Mean323.3016.8622.3410.1060.7900.3361.3852.101
SD17.800.9430.1930.0100.1250.0370.1710.105
N55555555
G3300Mean331.1716.8782.2260.1090.8100.3281.1992.023
SD24.040.8400.1040.0080.0570.0220.1700.035
N55555555
G41000Mean331.5115.3982.3920.1040.8340.3591.2972.034
SD18.731.3170.2220.0060.0580.0530.1510.043
N55555555

Key: Values are in Mean ± SD.



TABLE 27 (CONTD.) - SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS– FEMALES























































































































GroupsDose(mg/kg bw/day)Fasting Body Weight (FBW) and Absolute Organ Weights 
FBWOvariesUterusThyroid with Parathyroid
(g)(g)(g)(g)
G10Mean330.800.2290.6530.240
SD21.090.0220.0210.039
N10101010
G2100 Mean335.190.2430.6450.227
SD27.210.0380.0480.036
N10101010
G3300Mean336.320.2270.6500.254
SD24.610.0240.0450.046
N10101010
G41000Mean329.410.2270.6600.256
SD17.680.0200.0480.043
N10101010
       

Key: Values are in Mean ± SD.



TABLE 28 - SUMMARY OF RELATIVE ORGAN WEIGHTS – MALES























































































































































GroupsDose(mg/kg bw/day)Relative Organ Weights
LiverKidneysAdrenalsSpleenThymusHeartBrain
(%)(%)(%)(%)(%)(%)(%)
G10Mean3.0010.6530.0210.1920.1260.2830.418
SD0.1360.0820.0050.0220.0150.0120.051
N5555555
G2100 Mean2.9950.7120.0200.1970.1120.2990.414
SD0.0670.0530.0020.0240.0180.0170.035
N5555555
G3300Mean3.1050.6590.0190.2030.1290.321*0.426
SD0.1280.0450.0020.0190.0200.0230.045
N5555555
G41000Mean3.0300.7050.0210.1840.1110.324*0.403
SD0.2680.0530.0040.0140.0140.0250.019
N5555555

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05).


TABLE 28 (CONTD.)  - SUMMARY OF RELATIVE ORGAN WEIGHTS – MALES













































































































GroupsDose(mg/kg bw/day)Relative Organ Weights
TestesEpididymisProstate+ Seminal vesicles with Coagulating glandsThyroid with Parathyroid
(%)(%)(%)(%)
G10Mean0.7980.3360.6440.068
SD0.0720.0330.0850.008
N10101010
G2100 Mean0.7760.3100.6460.065
SD0.0580.0280.0540.009
N10101010
G3300Mean0.7870.3180.6560.062
SD0.0560.0460.0640.013
N10101010
G41000Mean0.7890.3280.6590.066
SD0.1200.0400.0700.011
N10101010

Key: Values are in Mean ± SD.


 


TABLE 29 - SUMMARY OF RELATIVE ORGAN WEIGHTS – FEMALES























































































































































GroupsDose(mg/kg bw/day)Relative Organ Weights
LiverKidneysAdrenalsSpleenThymusHeartBrain
(%)(%)(%)(%)(%)(%)(%)
G10Mean5.0710.7150.0310.2840.1010.3640.623
SD0.3380.0570.0020.0560.0050.0290.027
N5555555
G2100 Mean5.2250.7240.0330.2440.1040.4290.650
SD0.3590.0330.0040.0320.0100.0530.010
N5555555
G3300Mean5.1200.6750.0330.2460.0990.3630.613
SD0.4940.0590.0030.0290.0080.0560.039
N5555555
G41000Mean4.6470.7210.0310.2520.1080.3910.615
SD0.3370.0390.0030.0170.0120.0370.033
N5555555

Key: Values are in Mean ± SD.


 


TABLE 29 (CONTD.)  - SUMMARY OF RELATIVE ORGAN WEIGHTS – FEMALES































































































GroupsDose(mg/kg bw/day)Relative Organ Weights
OvariesUterusThyroid with Parathyroid
(%)(%)(%)
G10Mean0.0690.1980.073
SD0.0080.0140.012
N101010
G2100 Mean0.0750.1930.068
SD0.0150.0180.013
N101010
G3300Mean0.0680.1940.076
SD0.0100.0120.014
N101010
G41000Mean0.0690.2010.078
SD0.0070.0140.016
N101010

Key: Values are in Mean ± SD


 


TABLE 30 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – MALES                                                                                                                   





















































GroupG1G2G3G4
No. of Male Animals Observed10101010
 External Observations
NAD10/1010/1010/1010/10
 Internal Observations
NAD10/1010/109/1010/10
Testes(unilateral) – small sized0/100/101/10#0/10
Epididymis(unilateral) – small sized0/100/101/10#0/10

 Key: NAD = No Abnormality Detected:# = observed in the same animal (Animal No. 50).


 


Table 31 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – FEMALES







































GroupG1G2G3G4
No. of Females/Dams Observed10101010
External Observations
NAD10/1010/1010/1010/10
Internal Observations
NAD10/1010/1010/1010/10

 Key: NAD = No Abnormality Detected.


 


Table 32 - SUMMARY OF HISTOPATHOLOGY FINDINGS – MALE AND FEMALE (G1 AND G4)































Tissue/ Findings/SexMalesFemales
Dose GroupG1G4G1G4
Number Examined10101010
No Abnormality Detected10/1010/1010/1010/10

 


TABLE 32 (CONTD.): INDIVIDUAL ANIMAL HISTOPATHOLOGY FINDINGS - GROSS LESIONS





























Animal No.GroupSexOrgan/TissueGross FindingsHistopathology findings
50G3MaleTestes (unilateral)Small sizedAtrophy of seminiferous tubule, unilateral – 4D
Stage I to Stage XIV (PAS stain) – Absent, Unilateral
Epididymis (unilateral)Small sizedAspermia, unilateral

Note: Severity: - 1: Minimal; 2: Mild; 3: Moderate,4: Severe; Distribution: - F: Focal; M: Multifocal; D: Diffused; NAD: No Abnormality Detected

Conclusions:
Under the experimental conditions used in this study, the following effect levels were concluded for the test item:
• NOAEL for general toxicity was considered to be 1000 mg/kg bw/day in both genders, as no adverse effects on general health were observed in the study.
• NOAEL for reproductive toxicity (both genders) and maternal developmental toxicity was considered to be 1000 mg/kg bw/day, as no adverse effects on reproductive performance or maternal development were observed in the study.
• NOAEL for developmental toxicity in the offspring generation was considered to be 1000 mg/kg bw/day, as no adverse effects in the offspring generation were observed in the study.
Executive summary:

The purpose of this combined oral (gavage) repeated dose toxicity with the reproduction/developmental screening test in Wistar rats was to generate toxicological information concerning the effects of Phenethyl benzoate (CAS: 94-47-3) on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. The aim of the study was also to provide information on systemic toxicity, target organ toxicity, the No Observed Adverse Effect Levels (NOAELs), and the Lowest Observed Adverse Effect Levels (LOAELs).


One vehicle control (G1) and three graded dose levels of 100 (G2), 300 (G3) and 1000 (G4) mg/kg bw/day, with each group consisting of 10 males and 10 female rats with normal estrous cyclicity, were employed in the study. The test item, i.e. Phenethyl benzoate (CAS No. 94-47-3) in corn oil as vehicle, was given by oral gavage to animals of both sex at the doses of 100 (G2), 300 (G3) and 1000 (G4) mg/kg bw/day throughout the experiment period. Similarly, the vehicle (i.e. corn oil) was administered to the male and female rats in the vehicle control group (G1). Oral administration was done at a fixed dose volume of 5 mL/kg bw/day. Formulation analyses for dose concentration and homogeneity were performed twice during the treatment period. 


The males were dosed two weeks before mating, during mating, and after mating, for a total of 28 days. Females were dosed two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), during mating and gestation, and up to and including post-natal day (PND) 13 (i.e. up to the day before scheduled sacrifice on PND 14), for a total of approximately 58 days.


The following in-life and post-mortem examinations were performed in the adult male/female animals: mortality, morbidity, clinical signs of toxicity, detailed clinical signs of toxicity, body weight, food intake, oestrous cyclicity, functional observatory battery (functional and sensory reactivity examinations), pre-coital interval, mating index, fertility index, fecundity index, gestation length, gestation, index, live birth index, implantation sites, post-implantation loss, haematology, clinical chemistry (including thyroid hormone assessment), gross pathology, organ weight, and histopathology. For the offspring, the following in-life and post-mortem examinations were performed: litter size, litter weight, pup weight, sex ratio, anogenital distance (AGD) (including normalised AGD), nipple retention in male pups, pup survival, thyroid hormone levels, gross external and internal findings, organ weight (thyroid only), and histopathology (thyroid only).


 


Results


Morbidity and Mortality


No morbidity was observed at any dose level and all animals survived to scheduled sacrifice. 


Clinical Signs


No clinical signs of toxicity were observed at any dose level.


Body Weights / Body Weight Changes


No significant changes in body weight or body weight gain were observed at any dose level during the premating and mating period (male/females) or during the gestation and lactation period (females).


Food Consumption


No significant changes in food intake were observed at any dose level during the premating and mating period (male/females) or during the gestation and lactation period (females).


Functional Observational Battery


No significant changes in FOB parameters were observed, except for a statistically significant increase in the locomotor activity in terms of Z-movements in the G3 females (mean, 553 counts) compared to G1 females (mean, 484 counts). The above change was considered as incidental and therefore not toxicologically relevant.


Oestrous Cyclicity


The mean oestrous cycle length in G2 (4.10 days), G3 (4.03 days) and G4 (4.00 days) were comparable with G1 (mean, 4.05 days) and there were no significant changes in oestrous cycle pattern at any dose level.


Reproductive Performance and Development Indices


Male and female mating and fertility indexes, as well as female’s fecundity and gestation indexes were 100% in all dose groups. Live birth index was 100% in all groups. The number of dams conceiving between 1 - 5 days were 10/10, 9/10, 10/10 and 9/10 in G1, G2, G3 and G4, respectively and the number of dams conceiving between 6-14 days were 1/10 and 1/10 in G2 and G4, respectively. No significant changes in gestation lengths were observed between the groups. No significant changes in percent post implantation losses were observed in G2 (1.19 %), G3 (0.93 %) or G4 (0.97 %) compared to G1 (1.62 %).


Offspring data


No significant changes in litter size, litter weight, mean male pup body weight, mean female pups body weight, sex-combined pup weight, or sex ratio were observed in either G2, G3 or G4 when compared to G1. No significant changes in AGD or normalised AGD were observed at any dose level. No nipples/areolae were retained in any of the male pups on PND 12. Live birth index and pup survival indexes on PND 4 and PND 13 were 100% across all groups.


Thyroid Hormone Analysis


For adult male rats, no significant changes in T4 or TSH levels were observed at any dose level. In male pups, a significant increase in the TSH levels was observed in G2 (mean, 674.01 pg/mL) compared to G1 (564.25, pg/mL) and a significant decrease in T4 levels was observed in G4 (mean, 28.72 ng/mL) compared to the G1 (mean, 37.60 ng/mL). The observed effect on TSH levels in G2 was considered incidental due to lack of dose dependency. The observed effect on T4 in G4 could not be corroborated with any significant changes in thyroid weight, with any gross pathological findings of the thyroid, or with any histopathology findings of the thyroid (examined in G1 and G4) and was therefore considered to be non-adverse in nature.


Haematology


No significant changes in hematology were observed, except for a significant increase in WBC count in G3 males (mean, 8.94 × 103 cells/mm3) compared to G1 (mean, 6.80 × 103 cells/mm3) and a significant decrease in eosinophil count G3 males (mean, 0.60%) compared to G1 (mean, 2.00%). These changes in hematology in G3 males were not considered to be toxicologically relevant due to lack of dose dependency.  


Coagulation Potential Parameters


No significant changes in coagulation parameters were observed in the study.


Clinical Chemistry Parameters:


No significant changes in clinical chemistry were observed, except for a significant decrease in total cholesterol in G3 males (mean, 50.88 mg/dL) compared to G1 (mean, 69.94 mg/dL), a significant increase in ALT in G2 females (mean, 173.16 U/L) compared to G1 (mean, 124.87 U/L), and a significant increase in ALP in G2 females (mean, 199.33 U/L) compared to G1 (mean, 132.73 U/L). These changes in clinical chemistry in G3 males and in G2 females were not considered to be toxicologically relevant due to lack of dose dependency.  


Terminal Fasting Body Weights and Organ Weights


No significant changes in terminal fasting body weight were observed in the study. In terms of significant changes in absolute and relative organ weight, the follow changes were observed: a significant increase in absolute kidney weight in G2 males (mean, 3.885 gram) compared to G1 (mean, 3.379 gram), a significant increase in absolute heart weight in G4 males (mean, 1.760 gram) compared to G1 males (mean, 1.470 gram), a significant increase in relative heart weight in G3 males (mean, 0.321%) compared to G1 (mean, 0.283%), and a significant increase in relative heart weight in G4 males (mean, 0.324%) compared to G1 (mean, 0.283%). The significant increase in absolute kidney weight in G2 males was considered incidental due to lack of dose dependency. The significant increases in absolute and (or) relative heart weight in G3 and (or) G4 males could not be corroborated with any gross or histopathological findings of the heart in G4 and were therefore not considered to be adverse in nature. In pups, no significant changes in absolute thyroid with parathyroid weight were observed. 


Necropsy and Gross Pathology


Gross findings were limited to one incidence of unilateral small sized testes and epididymides in one male rat in G3 (Animal No. 050). This finding was considered to be spontaneous in nature and not toxicologically relevant due to lack of dose dependency.


There were no gross pathology findings in any of males and female pups sacrificed on PND 4 and 13.


Histopathology


Microscopic findings were limited to one incidence of aspermia (unilateral) in the epididymides and atrophy of the seminiferous tubules (unilateral, diffuse, severe), with complete absence of stages of spermatogenesis in the PAS-stained testes slides of the affected area, in one male rat in G3 (Animal No. 050). Tubular degeneration/atrophy in testes and aspermia in epididymides at a low incidence can be considered as a background finding in male rats (see Toxicologic Pathology, 40: 40S-121S, 2012 and “Background lesions in laboratory animals: a Color atlas” edited by EF McInness, Saunders Elsevier, Edinburgh, 2011, page 102, ISBN: 978-0-7020-3519-7). In due to this, and considering lack of dose dependency, the observed microscopic findings in the male rat in G3 (Animal No. 050) was considered to be incidental in nature and not toxicologically relevant. The stage of estrous cycle of all the adult females in G1 and G4 groups at the terminal sacrifice were in correlation with the histopathology of respective female reproductive organs. No histopathological findings were observed in the thyroid and parathyroid from any of the pups on PND 13.


Formulation analysis


Dose formulation analysis were performed at first week of pre-mating and during last week of lactation periods. Concentrations of the top, mid and bottom layers of all dose formulation samples were within the acceptable range of ±15 % to the nominal concentration (from 97.12% to 106.69%) with less than 10% relative standard deviations (from 0.78% to 6.25%). Therefore, all dose formulations prepared in this study were considered to be accurate, precise and homogeneous.


Conclusion:


Under the experimental conditions used in this study, the following effect levels were concluded for the test item:



  • NOAEL for general toxicity was considered to be 1000 mg/kg bw/day in both genders, as no adverse effects on general health were observed in the study.

  • NOAEL for reproductive toxicity (both genders) and maternal developmental toxicity was considered to be 1000 mg/kg bw/day, as no adverse effects on reproductive performance or maternal development were observed in the study.

  • NOAEL for developmental toxicity in the offspring generation was considered to be 1000 mg/kg bw/day, as no adverse effects in the offspring generation were observed in the study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The substance is not regarded to be classified for STOT RE as per Regulation EC 1272/2008, since no adverse effects on general health, in either male or female rats, were observed in the OECD 422 study with the substance following oral gavage exposure at 0, 100, 300 and 1000 mg/kg bw/day.