Dibutyl phosphonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 15th to August 10th, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From October 19th, 2020 to January 7th, 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Dose range finding study for the 90-day main study

Reason / purpose for cross-reference:

other: Validation of method used for the quantification of the substance

Reason / purpose for cross-reference:

other: Main study 90-days

Principles of method if other than guideline:

The objective of this study is to determine the adverse effect occurring as a result of the repeated daily oral administration of the Dibutyl Phosphonate to Wistar rats for a period of at least 28 consecutive days. Results of this study will be used for the dose selection of the 90-day oral repeated dose toxicity study according to the OECD 408 Guideline.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rat is selected as a test system because it has been historically shown to be a suitable model for repeated dose toxicity studies and is recommended in OECD Test Guidelines. The ECHA, EFSA EMA, EPA, FDA and other regulatory authorities also recommend it. Wistar strain has been chosen for this research because of its widespread use as a rodent model in toxicology studies and well-established historical information available about its genetic and physiologic background. Therefore, Wistar rat is selected as the test system to study the effect of the test item. The results of the research may be of value in predicting the toxicity of the test item to human being.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species/strain: Wistar rats, RccHan: WIST
- Source: Animal Breeding Facility (ABF) of testing laboratory
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old
- Housing: groups of 2-3 rats/sex/cage in sterilised solid floor polypropylene rat cages. Each cage was fitted with a stainless-steel top grille.
- Diet: Teklad Certified Global 14% Protein Rodent Diet, ad libitum.
- Water: clean and filtered drinking water (RO water filtration system), ad libitum
- Acclimation period: a total of 24 male and 24 female rats were received in the experimental room and acclimatised for a period of 5 days prior to randomisation.
- Duration of pre-treatment period: 2 days
- Health check: During acclimatisation, rats were were observed twice daily for clinical signs, mortality, and morbidity. On the day of randomisation, rats were weighed and observed for clinical signs, mortality, and morbidity. During the pre-treatment period, rats were observed twice daily for mortality, morbidity, and clinical signs.

DETAILS OF FOOD AND WATER QUALITY:
Quality of feed (microbial contaminant) and water (microbial and chemical contaminant) has been checked.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-23 (°C)
- Humidity: 65-66 (%)
- Air changes: 21 air changes per hour
- Photoperiod: 12 h light and 12 h dark; light hours were 06.00 - 18.00 h which was maintained by fluorescent tube lights attach to an autotimer.
- Light intensity: 150-162 lux

Route of administration:

oral: gavage

Details on route of administration:

Dose formulation was administered using intubation cannula attached to a graduated syringe. A constant dose volume of 10 mL/kg b. wt. was used, and individual dose was adjusted according to the most recently recorded body weight of each rat.

Vehicle:

corn oil

Details on oral exposure:

Dose Formulation Preparation
The test item was mixed with the corn oil to prepare dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing and with cannula intermittently during dosing. The dose formulations were prepared on each day of the dosing and was administered to rats immediately, after preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability, active ingredient (a.i.) concentration, and homogeneity of dose formulations were
analysed using a validated analytical method. Based on stability results, the test item was found stable up to 4 days in dose formulation.

Two sets of samples (10 samples per set) were collected by sampling three aliquots (upper, middle, and lower layer) from each concentration except from vehicle control (only one aliquot from middle layer). The sampling was done before initiation of the dosing on days 1 and 28, for the dose formulation analyses. One set of samples was used for analyses and the other set of samples was stored in refrigerated condition as a backup. The mean concentration for each dose formulation was determined and compared to the nominal value and the coefficient of variation was calculated. The acceptance criteria used for analysis was ±10% from nominal value (identified as “mean recovery” for a.i. concentration) and % CV < 10 (for homogeneity).

Duration of treatment / exposure:

The duration of dosing was 28 consecutive days. The first day of dose administration was designated as day 1 for each rat.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Dose Levels Selection
Three dose levels (low dose - 100, mid-dose - 500, and high dose - 1000 mg/kg b. wt./day) were selected based on available toxicity study data of test item. The available LD50 of acute oral toxicity study is > 3000 mg/kg b.wt. During the study, only one animal out of ten died in the 14-day post dosing observation period. In the lower dose, i.e., 1000 mg/kg b.wt. no mortality was observed.

Observations and examinations performed and frequency:

HOME CAGE OBSERVATIONS
Rats were observed for posture and presence or absence of convulsions.

HANDLING OBSERVATIONS
After completion of home cage observations, each rat was picked up by the observer and observed for below mentioned parameters: Ease of removing rat from cage, Handling, reactivity, Palpebral closure, Lacrimation, Eye examination, Piloerection, Skin examination and Salivation.

DETAILED CLINICAL OBSERVATIONS: yes
Mortality and Morbidity checks were performed twice daily. Clinical observations performed twice a day.

OPEN FIELD OBSERVATIONS
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 on it and observed for a period of 2 minutes. Each rat was observed for below mentioned parameters: Gait, Mobility, Arousal, Vocalisations, Rears, Respiration, Clonic movement, Tonic movement, Urination, Defecation, Stereotypy, Bizarre behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of rats from all groups were recorded at beginning of the treatment and at weekly intervals, thereafter. Rats from all groups were also weighed on the day of necropsy (fasted body weight). Body weight change compared to pre-treatment (day 1) body weight was calculated using below formula:
Body weight change (%) = Body weight on week (g) − Pre-treatment body weight (g) / Pre-treatment body weight (g) × 100

FOOD CONSUMPTION:
A weighed amount of feed offered, and the leftover feed was measured for each cage at weekly intervals, throughout the experiment. The food consumption was calculated and reported as g/rat/day using below mentioned formula:
Food consumption (g/rat/day) = Feed input (g) − Feed leftover (g) / Number of rats per cage × Number of days

FOOD EFFICIENCY: no

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no

OPHTHALMOSCOPIC EXAMINATION: no

HAEMATOLOGY: yes
- Time schedule for collection of blood: at the end of the treatment period
- Anaesthetic used for blood collection: yes (isoflurane anaesthesia by orbital plexus puncture)
- Animal fasted: yes
- Parameters examined: Haematocrit, Haemoglobin, Mean corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Mean corpuscular volume, Platelets count, Erythrocyte Count, Total Leukocyte Count, Reticulocyte Count, Prothrombin Time, Activated Partial thromboplastin Time.

CLINICAL CHEMISTRY: yes
Parameters examined: Alanine Aminotransferase, Albumin, Albumin/Globulin ratio, Alkaline Phosphatase; Aspartate Aminotransferase, Bile acids, Blood Urea Nitrogen, Calcium, Creatinine, Cretaine Kinase, Gamma Glutamyl Transpeptidase, Globulin, Glucose, High Density Lipoprotein, Inorganic Phosphorous, Lactate Dehydrogenase, Low Density Lipoprotein, Total Cholesterol, Total Protein, Total Protein, Total Bilirubin, Triglycerides, Urea, Chloride, Potassium, Sodium.

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: yes
To assess the behavioural and neurological status of each rat, NBO parameters were evaluated prior to initiation of the dosing and at weekly intervals, thereafter.

IMMUNOLOGY: no

Sacrifice and pathology:

GROSS PATHOLOGY
At terminal sacrifice (on day 29), rats were euthanised by carbon dioxide asphyxiation. All rats were subjected to a full gross necropsy under the supervision of a veterinary pathologist. Rats were examined carefully for external abnormalities. The cranial, thoracic, and abdominal cavities were cut, opened, and a thorough examination of organs was carried out to detect abnormalities.

ORGAN WEIGHT
Organs viz., adrenals, liver, kidneys, testes, epididymides, prostate + seminal vesicles and coagulating glands as a whole, thyroid with parathyroid, uterus with cervix, ovaries, thymus, heart, brain, and spleen, as applicable, from male and female rats were collected, weighed, and preserved. Adherent adipose tissue from organs was trimmed off and the wet weight of organs was recorded. The paired organs was weighed together, and combined weight was presented.

The thyroid with parathyroid weight was determined after fixation. All above mentioned organs including GI tract were preserved in 10% neutral buffered formalin solution. The organ weight ratios as percentage of body weight were determined. The microscopic examination was not performed, as treatment-related lesions and alterations in weights were not observed. Stomach was also not examined as lesions observed have not human corelevance.

Statistics:

Data were processed to get group means and standard deviations with significance between the vehicle control and treated groups. Parameters characterised by continuous data such as body weight, body weight change, food consumption, haematology parameters, clinical chemistry parameters, organ weight, and relative organ weight were subjected to Shapiro-Wilk’s test for normality followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s test (Gad, S.C. and Weil, C.S., 2007). When data did not meet the normality, data were transformed (log form and square root) to check the normality again. When, transformed data do not meet the normality, non-parametric tests were performed to calculate significance on original data. NBO parameters (urination, defecation, and rear) were subjected to non-parametric analyses. When data did not meet the homogeneity of variance, F-test was performed followed by t-tests to calculate significance. All parametric and non paramentric tests were performed using in-house developed and validated statistical software. The significance was calculated at the 5% and 1% levels for parametric tests and at the 5% level for non-parametric test between vehicle control and treated groups.



(Gad, S.C., and Weil, C.S., 2007: “Statistics for Toxicologists”, In Principles and Methods of
Toxicology, 5th Edition, Hayes A.W. (Ed), Raven Press Ltd., New York, p. 221-274)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rats from all groups were found normal during the observation period except salivation was observed during post dosing observation in male (from day 10 to 28) and female (from day 12 to 28) rats of mid dose group and also in male (from day 4 to 28) and female (from day 8 to 28) rats of high dose group. Salivation was started approximately 5 minutes post dosing and lasted until approximately 15 to 20 minutes post dosing. Salivation observed in rats was attributed to a response or physicochemical properties of the dose formulation and considered as non-adverse in nature.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight and mean body weight change of male and female rats from treatment groups were comparable with that of the vehicle control group except following a few statistical variations. Statistically significant decrease in the mean body weight change was observed during week 4 in male rats of the mid dose group when compared with that of vehicle control group. Statistically significat increase in the mean body weight change was observed during week 1 in male rats of the high dose group.
The marginal decrease in the body weight change of male rats was observed only at one interval and that not supported by the statistical decrease in the body weight and food consumption at same interval. Hence, it could not be considered as a test item treatment related effect

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of male and female rats from treatment groups was comparable with that of the vehicle control group

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Test item treatment did not lead to any alterations in haematological and coagulation parameters

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increase in ALT was observed in male rats of the high dose (G4) group. Increase without statistical significance was also noted in the high dose (G4) group female rats. Moreover, values of the high dose group rats (all male rats and 2 female rats) were above 95 percentile historical range (Male: 30.88 to 65.40, N = 240, Female: 20.60 to 62.05, N = 240). The effect was considered related to the test item treatment.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increase in rearing count was observed: during week 1 and week 3 in male rats of low dose group; during week 1 in male rats of mid dose group and during week 4 in female rats of high dose group.

Statistically significant increase in urination count was observed: during week 1 in male rats of low and high dose groups; during week 1 in female rats of low dose group.
Variation observed in rearing and urination count were dose independent and inconsistent. Hence, it could not be considered as effect of test item treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increase in the relative weight of the liver was observed in female rats of high dose (G4) group when compared with that of vehicle control group (G1). Effect without statistical significance was also noted in the male rats of the high dose group (G4) and considered as related to the test item treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

External examination of male and female rats from vehicle control and treatment groups did not revealany abnormality. Internal examination revealed increased thickness of non-glandular stomach in mid dose group (4/5 males and 3/5 females) and in all rats of high dose group. It was related to the test item treatment. However, this lesion is not relevant to human in absence of non-glandular stomach in human.

Description (incidence and severity):

HOME CAGE OBSERVATION:
Rats from the treatment and vehicle control groups showed normal posture viz., asleep (curled up often asleep), sitting B (sitting normally, feet tucked in), sitting C (sitting or standing alert, watching), and rearing. Clonic and tonic convulsions were absent in the home cage during NBO.

HANDLING OBSERVATIONS:
NBO performed during handling of rats did not reveal any abnormality. Rats revealed normal
behaviour during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). Eyelids were wide open in all rats. No rat showed lacrimation, salivation, and piloerection. Eye and skin examination did not reveal any abnormality in any rat.

OPEN FIELD OBSERVATIONS:
All rats from treatment and vehicle control groups showed normal gait, mobility, arousal and respiration during the ‘two-minute observation period’. Clonic and tonic movements, vocalisation, stereotypy, and bizarre behaviour were absent in this assessment. Rearing, urination and
defaecation count in male and female rats of treated groups were comparable with that of vehicle
control group except following a few statistical variation in rearing and urination count.
Statistically significant increase in rearing count was observed during week 1 and week 3 in male rats of low dose group when compared with that of vehicle control group. Similarly, statistically significant increase in rearing count was observed during week 1 in male rats of mid dose group and during week 4 in female rats of high dose group when compared with that of vehicle control group. Statistically significant increase in urination count was observed during week 1 in male rats of low and high dose groups when compared with that of vehicle control group. Similarly, statistically significant increase in urination count was observed during week 1 in female rats of low dose group when compared with that of the vehicle control group.
Variation observed in rearing and urination count were dose independent and inconsistent. Hence, it could not be considered as effect of test item treatment.

Remarks on result:

not measured/tested

Remarks:

Dose range finding study used to determine the doses to be used in the main study

Critical effects observed:

not specified

Active Ingredient Concentration and Homogeneity Analysis Results of a.i. concentration and homogeneity of formulated dose samples for low, mid, and high doses:

Sample Collected on Day	Concentration (mg/mL)
	Low dose - 10		Mid dose - 50		High dose - 100
	% Mean Recovery	% CV	% Mean Recovery	% CV	% Mean Recovery	% CV
1	103.06	3.63	96.82	0.57	98.26	1.82
28	100.88	0.43	99.49	0.68	100.19	1.12

The a.i. concentration and homogeneity of test item in dose formulation were within the allowable limit of ± 10 % of nominal concentration and % CV < 10.

Conclusions:

Based on the results, it is concluded that the test item produced effects at mid dose and high dose. The dose levels (mg/kg b. wt./day) prososed for 90-days toxicity study of test item are: 0-100-250-500 mg/kg bw/day

Executive summary:

Aim of the study was to determine the adverse effect occurring as a result of the repeated daily oral administration of the test item to Wistar rats for consecutive 28 days. The evaluation was conducted according to OECD 408.
The prepared dose formulations of test item in corn oil were administered by oral gavage at three graduated dose levels. The following doses were evaluated:
Control (G1): 0 mg/kg bw/day
Low Dose (G2): 100 mg/kg bw/day
Medium Dose (G3): 500 mg/kg bw/day
High Dose (G4): 1000 mg/kg bw/day

Each of the 4 groups comprised 5 male and 5 female Wistar rats. Rats from a concurrent vehicle control group (G1) received vehicle alone.

During the experimental period, rats were observed for signs of toxicity, morbidity, and mortality. Body weight was recorded for each rat, and food consumption was calculated for each cage, at weekly intervals. Neurobehavioral observations were performed at weekly intervals. At the end of the treatment period, clinical pathology estimations were performed in all rats. Rats were sacrificed and subjected to gross pathological examination. Weight of selected organs were recorded for all rats.

All parametric and non paramentric tests were performed using in-house developed and validated statistical software.

Results

The a.i. concentration and homogeneity results of dose formulation samples collected on days 1 and 28 were within acceptable range of ±10% of nominal concentration and %CV < 10.
No mortality or morbidity was observed in male or female rats of any group, throughout the study period.
Rats from all groups were found normal during the study period except salivation was observed during post dosing observation in male (from day 10 to 28) and female (from day 12 to 28) rats of mid dose group and also in male (from day 4 to 28) and female (from day 8 to 28) rats of high dose group. Salivation observed in rats was attributed to a response or physicochemical properties of the dose formulation and considered as non adverse in nature.
No treatment related change was observed in neurobehavioural observation parameters of treated rats.
Mean body weight, percent body weight change and food consumption of rats (male and female) from treatment groups were comparable with that of the vehicle control group.
Test item treatment did not lead any alteration in haematological and clinical chemistry parameters except increase in ALT level was observed in high dose group (G4) rats. Range of ALT level was higher than laboratory historical range, hence, it could be considered as effect of test item treatment.
Test item treatment did not lead any alteration in organ weight except increase was recorded in relative weight of the liver in high dose group (G4) rats and it was considered as effect of test item treatment.
External examination of male and female rats from vehicle control and treatment groups did not reveal any abnormality. Internal examination revealed increased thickness of non-glandular stomach in mid dose group (4/5 males and 3/5 females) and in all rats of high dose group. It was related to the test item treatment. However, this lesion is not relevant to human in absence of non-glandular stomach in human.

Based on the results, it is concluded that the test item produced effects at mid dose and high dose. The dose levels (mg/kg b. wt./day) prososed for 90-days toxicity study of test item are: 0-100-250-500 mg/kg bw/day

Cross-reference 2

Reason / purpose for cross-reference:

other: Validation method used for the quantification of the substance

Cross-reference 3

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted June 25th, 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl phosphonate

EC Number:

217-316-1

EC Name:

Dibutyl phosphonate

Cas Number:

1809-19-4

Molecular formula:

C8H19O3P

IUPAC Name:

dibutyl phosphonate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan: WIST

Details on species / strain selection:

Wistar rat is selected as a test system because it has been historically shown to be a suitable model for repeated dose toxicity studies and is recommended in OECD Test Guidelines. Wistar strain has been chosen for this research because of its widespread use as a rodent model in toxicology studies and well established historical information available about its genetic and physiologic background

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility (ABF) of the laboratory
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks old
- Weight at study initiation:
- Housing: Rats were housed in groups of 2 rats/sex/cage during the study period in sterilised polypropylene rat cages while one male rat having more than 500 g body weight was housed individually in a separate cage after completion of 17 weeks of treatment. Each cage was fitted with a stainless steel top grille having provision for keeping rodent pellet feed and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks and cage rotation was performed at weekly intervals in each cage rack which ensures almost similar environmental conditions for rats from different groups. The bottom of the cage was layered with clean sterilised paddy (rice) husk as the bedding material. Rats were provided wooden chew blocks as an environmental enrichment material in each cage. Cages, bedding, and enrichment materials were changed alternate day throughout the study period. Cage racks were cleaned daily with cloth soaked with disinfectant solution and water bottles were changed daily, throughout the study period. Cage lids were changed once every two weeks throughout the study period. Contaminant analysis (chemical and microbial) of the bedding and enrichment material samples are routinely performed
- Diet: ad libitum, standard rodent pellet feed ad libitum (Teklad Certified Global 14% Protein Rodent Diet, Envigo, USA)
- Water: unlimited supply of clean and filtered drinking water (RO water filtration system) in autoclaved polypropylene bottles
- Acclimation period: 5 days prior to randomisation. During the acclimatisation period, all rats are marked with a temporary identification number on the base of the tail using a marker pen and were observed twice daily for a clinical signs, morbidity, and mortality.
- Other: Every day, the floor of the experiment room was cleaned and mopped with 1% v/v gramicid surface disinfectant solution. All worktops were also cleaned with cloth wetted with the same disinfectant solution.

DETAILS OF FOOD AND WATER QUALITY: Every feed consignment received is accompanied by a certificate of analysis of nutrient content with chemical contaminant from the supplier. Quality of feed (microbial contaminant) and water (microbial and chemical contaminant) is being checked regularly at six-month intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 65-67
- Air changes: minimum of 15 per hour.
- Photoperiod: 12 h light and 12 h dark
- Light intensity: fluorescent tube lights using an auto timer; 159-228 LUX
- The experimental room temperature and relative humidity were recorded daily, while light intensity and air changes were recorded monthly.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Dose formulation will be administered once daily orally through gavage using intubation cannula
attached to a graduated syringe. A constant dose volume of 10 mL/kg b. wt. will be used, and individual dose will be adjusted according to the most recently recorded body weight of each rat. Rats from the vehicle control groups will be given the vehicle alone.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was mixed with the corn oil to prepare dose formulations of required concentrations. The prepared dose formulations were thoroughly mixed using a magnetic stirrer before dosing and with cannula intermittently during dosing. The dose formulations were prepared on each day of the dosing and was administered to rats immediately, after preparation.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was selected as a vehicle based on solubility check. On the basis of the stability results, the test item was found to be stable up to 4 days in dose formulation at room temperature.

TEST ITEM ADMINISTRATION
Dose formulation was administered once daily orally through gavage, using intubation cannula attached to a graduated syringe. A constant dose volume of 10 mL/kg b. wt. was used, and individual dose was adjusted according to the most recently recorded body weight of each rat. Rats from the vehicle control group received the corn oil only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability, active ingredient (a.i.) concentration, and homogeneity of dose formulation were
analysed at the Department of Chemistry using a validated analytical method. Based on stability study results, the test item was found stable up to 4 days in dose formulation.

Instrumental parameters for dose formulation method of analyses:
Instrument : LC-MS/MS (4000 Qtrap Mass spectrometer coupled with Nexera X2 HPLC system) or Equivalent
Column : X-Select CSH, Fluoro phenyl [150 x 4.6 mm (i.d.), 3.5 μm particle size] or Equivalent
Flow Rate : 0.7 mL/Minute
Injection Volume : 2 μL
Cooler Temperature : 10 °C
Column Temperature : 40 °C
Mobile Phase : Methanol: 0.1% Formic acid in Milli-Q water (80:20 %), v/v

Two sets of samples (10 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from each concentration except vehicle control (only middle layer). The sampling was performed prior to initiation of dosing and at monthly intervals thereafter, for dose formulation analysis. One set of samples was used for analyses and other set of samples was stored in the refrigerator. The second set of samples will be disposed of, during report finalisation. The mean concentration was determined and compared to the nominal value and the coefficient of variation calculated. The acceptance criteria used for analysis was ±10% from nominal value and %CV < 10.

Duration of treatment / exposure:

90 days

28 days - recovery period (for vehicle control group and highest dose group)

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

90-day exposure period (G1)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

90-day exposure period (G2)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

90-day exposure period (G3)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

90-day exposure period (G4)

No. of animals per sex per dose:

ten per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Three dose levels (low dose - 100, mid-dose - 250 and high dose - 500 mg/kg b. wt.,/day) were selected based on the results of a 28 day dose range finding study
- Rationale for animal assignment: On the day of randomisation, rats were weighed and observed for clinical signs, mortality, and morbidity. During randomisation, 60 male and 60 female rats were selected for the study. Selected rats were randomly allocated to different groups using a validated computer software program. Rats were divided into four main groups and two recovery groups. Each group consisted of 10 male and 10 female rats. The remaining 6 male and 6 female rats were returned to the laboratory.
After randomisation, 60 male and 60 female rats were housed for 4 (male) to 5 (female) days pre-treatment period, under experimental room conditions before commencement of dosing. During the pre-treatment period, baseline NBO and opthalmological examination were performed on rats from all groups. Rats from all groups were also observed twice daily for mortality, morbidity, and clinical sign
- Fasting period before blood sampling for clinical biochemistry: overnight fasting
- Rationale for selecting satellite groups: Rats allocated to the recovery groups were maintained untreated for a period of 28 days, to assess reversibility, persistence or progression of any toxicity after the treatment for 90 days.
- Post-exposure recovery period in satellite groups: 28 days

Positive control:

none

Examinations

Observations and examinations performed and frequency:

MORTALITY AND MORBIDITY
All rats were observed twice daily for mortality and signs of morbidity.

DETAILED CLINICAL OBSERVATIONS
All rats were observed twice daily for clinical signs

BODY WEIGHT
- The body weight of all rats was recorded at the beginning of the treatment and weekly intervals, thereafter. Rats from all groups were also weighed on the day of necropsy (fasted body weight). The percentage body weight change compared to pre-treatment body weight was calculated

FOOD CONSUMPTION AND COMPOUND INTAKE:
- A weighed amount of feed was offered and leftover feed was measured in each cage at weekly intervals during the study period.

OPHTHALMOSCOPIC EXAMINATION
An ophthalmological examination was performed on each rat with the aid of ophthalmoscope - once before the commencement of the treatment and second time before sacrifice (at terminal and recovery). In order to facilitate easy examination of the anterior part of the eye, homatropine (2%) eye drops were instilled in to eye to dilate the pupil. This mydriatic solution was instilled into the eye approximately 15 to 20 minutes before the eye examination.

CLINICAL CHEMISTRY - HAEMATOLOGY
At the end of treatment and recovery periods, blood was collected from all rats under light isoflurane anaesthesia by orbital plexus puncture using a fine heparinised capillary tube. Rats were fasted overnight (with ad libitum supply of drinking water) prior to blood collection (approximately 3 mL blood). Blood samples were collected for haematology (in vials containing 4% EDTA anticoagulant for whole blood), coagulation parameters (in vials containing 3.2% sodium citrate anticoagulant for plasma separation), and clinical chemistry analysis (in plain vials for serum separation).
Serum thyroid hormones (T3 and T4) was analysed using the validated bioanalytical method and serum TSH was analysed by ELISA methods.
Plasma samples were stored at -70 ± 10 °C till the analysis of coagulation parameters while, analysis of haeamatology, clinical chemistry, and urine parameters was performed on the same day of sample collection.
- Haematology/coagulation parameters checked: Haematocrit, Haemoglobin, Mean Corpuscular Haemoglobin, Mean Corpuscular Haemoglobin Concentration, Mean Corpuscular Volume, Platelets Count, Erythrocyte Count (Red Blood Cells), Total Leukocyte Count (White Blood Cells), Differential Leukocyte Count, Reticulocyte Count, Prothrombin Time Activated Partial Thromboplastin Time
- Clinical chemistry parameters checked: Alanine Aminotransferase, Albumin, Alkaline Phosphatase, Aspartate Aminotransferase, Bile Acid, Calcium, Creatinine, Creatinine Kinase, Gamma Glutamyl Transpeptidase, Glucose, Inorganic Phosphorous, Total Cholesterol, Total Protein, Total Bilirubin, Triglycerides, Urea, Lactate Dehydrogenase, High-density lipoproteins, Low-density lipoproteins, Albumin: Globulin ratio, Globulin, Blood Urea Nitrogen, Chloride, Potassium, Sodium

URINALYSIS
Urine was collected individually from all rats at the end of treatment and recovery periods in urine collection tubes. For urine collection, rats were housed overnight in metabolic cages during which feed was withheld but the water was provided ad libitum.
- Parameters checked: Appearance, Colour, Volume, Sediment evaluation (Microscopic examination), Specific gravity, pH, Protein, Glucose, Ketone, Blood, Bilirubin, Urobilinogen

NEUROBEHAVIOURAL EXAMINATION
To assess the behavioural and neurological status of each rat, below mentioned parameters were evaluated prior to initiation of dosing and at least at weekly intervals thereafter.
- Home Cage Observations: In-home cage, rats were observed for posture and presence/ absence of convulsions. Observations were recorded using proper descriptions.
- Handling Observations: After completion of home cage observations, the observer picked up the rat and rank for ease of removal from the home cage and ease of handling reactivity. During handling, the rat was examined for palpebral closure, lacrimation, salivation, piloerection, eye, and skin abnormality, if any. Observations were recorded using proper rank/ description.
- 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 this period, the rat was observed for its gait abnormality, mobility score, arousal level, the occurrence of spontaneous or unprovoked vocalisations, rearing, ease of respiration, convulsions, number of urine pools and faecal boluses, and stereotypy and bizarre behaviour. Observations were recorded using a proper rank/ description.

FUNCTIONAL OBSERVATION BATTERY (FOB)
Following FOB parameters were performed during the 12th week of the treatment (G1 to G4) and the last week of the recovery period (G5 and G6) for rats.
- Motor Activity: Motor activity was evaluated for all rats using an automated photobeam activity system equipped with a validated computerised PAS software. 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 including total activity, ambulatory activity, and fine activity were evaluated.
- 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. Below mentioned parameters were performed and recorded for rats.
- Approach response: Rats were 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.
- 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 an absent, slight, normal, or exaggerated response
- Click response: A clicker was positioned approximately 5 cm above the back of the rat, ensuring not to have the clicker in the rat's field of vision. The clicker was held in the palm of the hand ensuring consistency of sound from test to test. The degree of the elicited response of the rat to the click sound was recorded as an absent, slight, normal, or exaggerated response.
- 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.
- 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.
- Air Righting Reflex: The rat was held supine, with the hands of the observer under the back and shoulders of the 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
- Grip Strength: Grip strength of both forelimb and hindlimb was measured with a grip strength meter to determine the ability of the rat to grasp and hold on the mesh platform. The grip strength of each rat was measured for 3 consecutive times; results were averaged separately for the forelimb and hindlimb.
- Foot Splay: The landing hind limb feet of each rat was marked with a non-permanent, non-toxic ink just prior to testing. The 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 thrice. The distance between the two foot prints was measured and an average of three-foot splay values was calculated.

ESTROUS CYCLE EVALUATION
On the day of necropsy, vaginal smears were examined in all surviving female rats to determine the stage of the oestrous cycle. Care was taken to avoid disturbance of mucosa while obtaining vaginal/cervical cells.

Sacrifice and pathology:

GROSS PATHOLOGY
At scheduled terminal and recovery sacrifices, all rats were euthanised by carbon dioxide asphyxiation and subjected to a full gross necropsy under the supervision of a veterinary pathologist. All rats were examined carefully for external abnormalities. The cranial, thoracic and abdominal cavities were cut, opened and a thorough examination of the organs were carried out to detect abnormalities.

ORGAN/TISSUE COLLECTION
The following organs and tissues, as applicable, from male and female rats of main and recovery groups, were collected, weighed, and preserved. Adherent adipose tissue from the organs was trimmed off and wet weights of organs were recorded. All organs were preserved in 10% neutral buffered formalin solution except the testes and eyes which were collected in modified Davidson’s fluid and Davidson’s fluid, respectively. The paired organs were weighed together and combined weights were presented. The thyroid with parathyroid and pituitary gland weight was determined after fixation. The organ weight ratios as a percentage of terminal body weight were determined.
- For organ weight: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Spleen, Seminal vesicles with coagulating glands and prostate, Thymus, Testes, Thyroid with parathyroid, Uterus with cervix
- For microscopic examination: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary gland, Spleen, Seminal vesicles with coagulating glands and prostate, Thymus, Testes, Thyroid with parathyroid, Uterus with cervix, Aorta, Bone with marrow (femur with a joint), Esophagus and trachea, Eyes, Gross lesions if any, Lungs, Lymph nodes – prescapular, Lymph nodes – mesenteric, Large intestine (cecum, colon, and rectum), Mammary glands, Pancreas, Stomach, Small intestine (duodenum, jejunum and ileum with Peyer’s patches), Skin, Sciatic nerves, Skeletal muscle, Salivary glands, The spinal cord at three levels - cervical, thoracic and Lumbar, Urinary bladder, Vagina

HISTOPATHOLOGICAL EXAMINATION
The histopathological examination was carried out for preserved organs and tissues of rats from vehicle control (G1) and high dose (G4) groups. In addition, all gross lesions were examined microscopically. All organs and tissue samples, as given above were processed, embedded, and cut at a thickness of 3 to 5 micrometers and stained with hematoxylin and eosin. The histopathology peer review was carried out. Histopathological examination revealed treatment related alterations in non-glandular stomach of high dose group therefore, stomach was processed from low, mid and recovery groups of both sexes.

Statistics:

Data were processed to get group means and standard deviations with significance between the control and treated groups, using validated statistical software. All parameters characterised by continuous data such as body weight, Body weight change, food consumption, FOB parameters (grip strength and foot splay), organ weight, relative organ weight, and clinical pathology (haematology, clinical chemistry, and some of the urinalysis parameters) were subjected to Shapiro-Wilk’s test (wherever applicable) for normality followed by Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s test (Gad, S.C. and Weil, C.S., 2007). When data do not meet the normality, data were transformed (log form, square root, etc.) to check the normality again. If transformed data do not meet the normality, non-parametric tests were performed to calculate significance. When the data do not meet the homogeneity of variance, F-test was performed followed by the t-tests to calculate significance. NBO parameters (urination, defecation, rear, and vocalisation) and FOB parameters (motor activity) data were subjected to non-parametric tests to calculate significance. The significance for Parametric was calculated at 5% and 1% level between vehicle control and treated groups, while for non-parametric was calculated at 5% level between vehicle control and treated groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All rats were normal, throughout the dosing period, in control, low, and mid dose groups. Mild salivation was observed in male and female rats of high dose treated groups.
Salivation started approximately 15 to 30 minutes post-dosing and lasted until approximately at 50 to 60 minutes post-dosing. Salivation was observed only during post-dosing observation and not seen in morning observation. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity (Wook-Joon Yu at al, 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful toxicological significance, since no related changes were observed in the low and mid dose groups

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity was observed in any group, throughout the treatment and recovery periods

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight and mean body weight change of male and female rats of treatment groups were comparable with those of respective vehicle control groups except following statistical variations:,
- Statistically, a significant decrease in the mean body weight change was observed during week 9, 10 and 11 in male rats of low dose group (G2) when compared with that of vehicle control group (G1).
- Decrease in the mean body weight change was inconsistent and dose independent. Also, these changes are limited to one sex only. Hence, it could not be considered as an effect of test item treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption of male and female rats of treatment groups were comparable with those of respective vehicle control groups except following statistical variations:
• Statistically, a significant decrease in the mean food consumption was observed during week 5, 8 and 13 in male rats of low dose group (G2) when compared with the vehicle control group (G1).
• Statistically, a significant decrease in mean food consumption was observed during week 10, 12 and 13 in male rats of mid dose group (G3) when compared with that of the vehicle control group (G1).
• Statistically, a significant decrease in mean food consumption was observed during week 12 and 13 in male rats of high dose group (G4) when compared with that of the vehicle control group (G1).
• Statistically, a significant decrease in mean food consumption was observed during week 7 and 13 in male rats of high dose recovery group (G6) when compared with that of the vehicle control recovery group (G5).
• Statistically, a significant decrease in mean food consumption was observed during week 4, 5, 12 and 13 in female rats of low dose group (G2) when compared with that of the vehicle control group (G1).
• Statistically, a significant decrease in mean food consumption was observed during week 4, 12 and 13 in female rats of mid dose group (G3) when compared with the vehicle control group (G1).
• Statistically, a significant decrease in mean food consumption was observed during week 4 and 13 in female rats of high dose group (G4) when compared with that of the vehicle control recovery group (G1).
• Statistically, a significant decrease in mean food consumption was observed during week 5 in female rats of high dose recovery group (G6) when compared with that of the vehicle control recovery group (G5).
Reduction in the mean food consumption was intermittent and dose independent. Also, body weight growth of these animals was not statistically significant. These changes could be due to the irritant properties of test item in gastric mucosa and were marginal. Hence, it could be considered as an effect of test item treatment but not adverse in nature.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmological examination of rats from all groups did not reveal any abnormality

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related alteration was noted in haematological parameters except following few statistical variations.
In prothrombin time (PT), a statistically significant decrease was noted in all treated main groups of males, while statistically significant increase was observed in mid dose (G3) and high dose (G4) females. Effect was not considered related to the test item treatment due to the inconsistency of the effect between sexes and because the values of all animals are within the historical range [Male (N = 315): 8.7 to 14.6; Female (N = 320): 8.3 to 13.0; 2.5th to 97.5th percentile].
In the eosinophil count, statistically significant decrease was noted in the high dose (G4) males, while the same was found increased significantly in high dose recovery (G6) males. Effects were not considered related to the treatment in absence of consistency between sexes . Similarly, statistically significant increase noted in the platelet in high dose recovery (G6) females was not considered related to the treatment in absence of consistency between sexes, and lack of the effect at the end of treatment in the G4 females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related alteration was noted in clinical chemistry except following few statistical variations.
A statistically significant decrease was noted in the AST, total protein, and globulin in low dose (G2) males, and total bile acids in G2 and mid dose (G3) males, while statistically significant increase was found in albumin:globulin ratio in G2 males and females. Alterations were not related to the test item treatment in absence of dose dependency.
At the end of recovery, statistically significant increase was noted in the serum triglycerides, ALT, and total bilirubin in high dose recovery (G6) males and glucose in G6 females. Effects were not related to the test item treatment in absence of consistency between sexes and values were within historical ranges [Male (N = 280) serum triglycerides: 34.88 to 161.18; ALT: 29.21 to 89.41; total bilirubin: 0 to 6.68; Female (N = 285): glucose: 83.45 to 186.94; 2.5th to 97.5th percentile]

Endocrine findings:

no effects observed

Description (incidence and severity):

Hormone Analysis
TSH analysis did not reveal any effect related to test item treatment in male and female rats of treatment and recovery groups. Statistically, significant decrease in the serum T4 level was observed in male rats of mid (G3) and high dose (G4) groups when compared with that of the vehicle control group (G1). Statistically, significant decrease in the serum T4 level was observed in male and female rats of high dose recovery group (G6) when compared with that of the vehicle control recovery group (G5). Statistically, significant increase in the serum T3 level was observed in male rats of high dose recovery group (G6) when compared with that of the vehicle control recovery group (G5).
These alterations were not considered likely to be related to the test item treatment in the absence of any histopathological effects in the thyroid and/or parathyroid, and because all values were within historical control range.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urine analysis parameters did not reveal any alteration related to treatment

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Neurobehavioural Observations (NBO)
- Home Cage Observations: In the home cage, all rats from the treatment (G2, G3, G4, and G6) and vehicle control groups (G1 and G5) showed normal posture, 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.
- Handling Observations: NBO performed during the handling of rats did not reveal any abnormality. All rats revealed a normal behaviour during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). No rat showed lacrimation, salivation , and piloerection. Eyelids were wide open in all rats. The eye and skin examination did not reveal any abnormality in any rat.
-Open Field Observations: In the open field, all rats from treatment and vehicle control groups showed normal gait, mobility, arousal and respiration during the two-minute observation period. Clonic and tonic movements, vocalisation, stereotypy, and bizarre behaviour were absent in this assessment. The rear, urination, and defecation counts of male and female rats from treatment groups were comparable with those of the respective vehicle control groups except, following statistical variations;
• Statistically, significant increase in rearing count was observed during week 5 in male rats of high dose group (G4) when compared with that of the vehicle control group (G1).
• Statistically, significant decrease in rearing count was observed during week 1 in male rats of high dose recovery group (G6) when compared with that of the vehicle control recovery group (G5).
• Statistically, significant increase in rearing count was observed during week 2 in female rats of high dose group (G4) when compared with that of the vehicle control group (G1).
• Statistically, significant increase in rearing count was observed during week 14 and 15 in female rats of high dose recovery group (G6) when compared with that of the vehicle control recovery group (G5).
• Statistically, significant increase in urination count was observed during pre-treatment in male rats of mid dose group (G3) when compared with that of the vehicle control group (G1).
These findings were intermittent and dose-independent. Hence, it could not be considered as toxicologically relevant.

Functional Observational Battery (FOB)
- Motor Activity: The motor activity data of rats from treatment groups were comparable with those of the respective vehicle control groups
- Sensory Reactivity Observations: Sensory reactivity parameters, viz. , approach response, touch response, click response, tail pinch response, pupil response, and air righting reflex in rats of treatment groups were comparable with those of the respective vehicle control groups.
- Grip Strength: The hindlimb grip strength value of rats of treatment groups was comparable with those of the respective vehicle control groups except statistically significant increase in hindlimb grip strength was observed in female rats of high dose recovery group. These variation in grip strength was not supported by any other neuromuscular findings and limited to one sex only. Hence, these findings were not considered as treatment-related.
-Foot Splay: Hindlimb foot splay value of rats from treatment groups was comparable with those of the respective vehicle control groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related change was observed in the terminal body weight, organ weight, and relative organ weight of rats from treatment groups except from the following few statistical variations.
Statistically significant decrease was observed in the absolute weight of ovaries in high dose (G4) females. It was more likely to be related to the cyclical change and not related to the test item treatment. Moreover, histopathological alteration was not observed in the ovaries of high dose group.
After the recovery period, statistically significant increase was noted in absolute and relative weight of liver (9% and 6% increase than vehicle control, respectively) and absolute weight of kidneys (7% increase than control) in high dose recovery (G6) females. Effects were not considered as related to the test item treatment due to the absence of similar alterations at terminal sacrifice and due to the lack of consistency between sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Internal gross examination revealed increase in thickness of non-glandular stomach in all 3 treated main groups of both sexes (except in G2 males) in dose dependent manner. Lesion was considered as related to the test item treatment and found recovered completely after the recovery period. However, the change was considered as insignificant due to the lack of human relevance as non-glandular stomach is absent in humans.
Testes and epididymides examination revealed reduced size in one animal each from low dose (G2), mid dose (G3) and vehicle control recovery (G5), which was considered as incidental/ spontaneous and not related to the test item treatment

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed hyperkeratosis and mucosal hypertrophy/hyperplasia in non-glandular stomach in all treated main groups of both sexes in dose dependent manner. Lesions were related to the test item treatment but considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans (Jeff Mckee, 2011). Marked recovery was observed in the effects after the recovery period.
In females, additionally, mucosal degeneration and submucosal MNC infiltration were also noted as treatment related lesions in G4. The incidence of these lesions was very low and not observed in recovery group animals .
The rest of the microscopic lesions observed in various organs were at a lower rate of occurrence and were mostly minimal to mild in nature, non-specific and insignificant. Hence, these lesions were considered as incidental or physiological and not treatment related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous Cycle Status of Termination
The test item did not show any adverse effects on the estrous cycle of female rats at any dose level. The pattern and number of females showing estrous cycle stages indicates the normal cyclicity of females in all study groups (G1 to G6). This was further supported by normal histopathlogical observations in the uterus, cervix and vagina of G4 females.

Effect levels

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects relevant to humans identified up to the highest dose administered

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Active Ingredient Concentration and Homogeneity Analysis

Sample Collected on Day	Concentration (mg/mL)
	Low dose - 10		Mid dose - 25		High dose - 50
	% Mean Recovery	% CV	% Mean Recovery	% CV	% Mean Recovery	% CV
1	99.73	1.43	100.60	0.50	100.37	1.41
29	105.74	1.00	104.42	0.16	104.65	0.89
57	108.60	3.00	111.21	0.75	109.69	1.93
85	109.58	1.00	106.16	8.41	113.53	1.73

The a.i. concentration and homogeneity of the test item in dose formulation was within allowable limit of ±10% of the nominal concentration except on day 57 for mid dose group and on day 85 for high dose group where the recovery was slightly higher than normal range i.e., 1 to 3% and %CV < 10. Higher recovered concentration could be due to slight immiscibility of test item in corn oil and also that was within ±15% which is acceptable for such mixtures.

Applicant's summary and conclusion

Conclusions:

NOAEL (rat, male/female) = 500 mg/kg bw/day

Executive summary:

This study was conducted to evaluate the potential toxicity of dibutyl phosphonate in Wistar rats following daily oral gavage administration for 90 consecutive days. Four groups of 10 female and 10 male rats each were exposed at 0, 100, 250 and 500 mg/kg bw/day for 90 days. A recovery period of 28 days for the vehicle control and the highest groups also run. The vehicle chosen was corn oil. 

Dose formulation analysis was performed in order to evaluate the homogenicity and the active ingredient concentration. The formulations were prepared every day and were administred shortly after their preparation. During the experimental period, all rats were observed for signs of toxicity, morbidity, and mortality. Body weight was recorded at weekly intervals. Body weight change and food consumption were calculated at weekly intervals. An ophthalmological examination was performed before treatment and sacrifices. Neurobehavioral observations were performed at weekly intervals. Functional observational battery was performed during 12^th week in main groups and during 4^th week in recovery groups. At the end of the treatment and recovery periods, clinical pathology estimations were performed in all rats. Vaginal smears was examined on the day of necropsy in all female rats. At the end of the treatment and recovery periods, all rats were sacrificed and subjected to gross pathological examination. The weight of selected organs was recorded for each rat. Microscopic examination of tissues and organs was performed in vehicle control (G1) and high dose (G4) groups. Histopathological examination revealed treatment related alterations in non-glandular stomach. Therefore, stomach was processed from low, mid, and recovery groups of both sexes.

Results

Mortality and morbidity: No mortality or morbidity was observed during the treatment and recovery periods.

Clinical Observations: All rats were normal, throughout the dosing period, in control, low, and mid dose groups. Mild salivation was observed during post dosing observation in male and female rats of high dose treated group. It is well known that salivation is often observed in gavage studies and may be a reaction to the taste or irritation of the test article rather than an indication of toxicity (Wook-Joon Yu at al, 2011). Therefore, it was considered that the transient salivation observed in this study was of doubtful toxicological significance, since no related changes were observed in the low and mid dose groups.

Ophthalmological examination: Ophthalmological examination of all rats did not reveal any abnormality.

Neurobehavioural Observations: No treatment-related change was observed in neurobehavioural observations performed in male and female rats from treatment groups.

Functional Observational Battery: No treatment-related change was observed in functional observational battery parameters performed in male and female rats from treatment groups

Body Weight and Body Weight Change: No treatment-related change was observed in body weight and percent body weight change in rats from treatment groups when compared with those of respective vehicle control groups.

Food Consumption: Intermittent reduction in the mean food consumption was observed in male and female rats of treatment groups. Also, body weight growth of these animals were not that much impacted. These changes could be considered due to irritant properties of test item in gastric mucosa and were marginal. Hence, it could be considered as effect of the test item treatment but not adverse in nature.

Clinical pathology: No treatment-related change was observed in haematology, coagulation, clinical chemistry, and urinalysis parameters of rats from treatment groups when compared with those of respective vehicle control groups.

Organ weight: No treatment-related change was observed in the terminal body weight, organ weight, and relative organ weight of rats from treatment groups when compared with those of respective vehicle control groups.

Macroscopic examination: Internal gross examination revealed increase in thickness of non-glandular stomach in all 3 treated main groups of both sexes (except in G2 males) in dose dependent manner. Lesion was considered as related to the test item treatment. The change was considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans.

Microscopic examination: Microscopic examination revealed hyperkeratosis and mucosal hypertrophy/hyperplasia in non-glandular stomach in all treated main groups of both sexes in dose dependent manner. Lesions were related to the test item treatment and considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans (Jeff Mckee, 2011). Marked recovery was observed in the effects after the recovery period.

Conclusion:

There was no treatment-related change observed in in-life phase parameters except the clinical signs like salivation in male and female rats treated with 500 mg/kg b. wt. and intermittent decrease in food consumption in male and female rats treated with 100, 250 and 500 mg/kg b. wt. These changes were not having toxicological significance in this study and considered as non-adverse in nature.

Haematology, coagulation, urine analysis and organ weights parameters did not reveal any test item treatment related change.

On gross examination, treatment revealed thickness of non-glandular stomach in all treatment groups, which was associated with histopathological lesions. The change was considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans.

Hyperkeratosis and mucosal hypertrophy/hyperplasia in non-glandular stomach was observed in all treated main groups of both sexes in dose dependent manner and considered as insignificant due to lack of human relevance as non-glandular stomach is absent in humans.

Therefore, the No Observed Adverse Effect Level (NOAEL) of dibutyl phosphonate is 500 mg/kg b. wt./day.