Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

dimethoxyphosphinic acid; methoxyphosphonic acid; phosphoric acid

EC Number:

908-996-7

IUPAC Name:

dimethoxyphosphinic acid; methoxyphosphonic acid; phosphoric acid

Test material form:

other: liquid

Details on test material:

Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate
EC No.: 908-996-7

Specific details on test material used for the study:

OURCE OF TEST MATERIAL
- Source of test material: Clariant
- Batch No.of test material: DEH8 006695
- Expiration date of the batch: 30.10.2019
- Date of manufacturing: 31.10.2017
- Purity as per Certificate of Analysis: 99.95%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigeration ((+15 to +25°C)
- Stability under test conditions: The concentration and stability of the test item in the vehicle were established at 1 and 100 mg/mL concentrations under Eurofins Advinus Study No. G16490. Based on the results, the test item was stable and homogenous in the vehicle up to 5 days when stored at room temperature

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 20 to 24°C and relative humidity was between 56 to 68 %. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour was maintained in the experimental room.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once orally for 90 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 28 days following the 90-day treatment period.
The dose formulation and the vehicle were administered at an equivolume of
10 mL/kg/day. The dose volume was calculated for individual animals on the first day of treatment and was adjusted according to the most recent body weights recorded during the treatment period

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 33) and 3rd (Day 63) month of the treatment period and analysed in-house. For each set, six composite samples were drawn from each preparation and in case of control, duplicate samples were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G16490. One set of samples were analyzed for concentration analysis. On day 1, G2 (5 mg/mL) and G4 (30 mg/mL) concentration samples were out of specification, could be due to sample processing error. Hence, the analysis was repeated using second set of samples.
Formulations were considered acceptable as overall mean results of all samples are within ± 15 % of the theoretical concentration and the overall relative standard deviation (RSD) was less than 10 %.

Duration of treatment / exposure:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once orally for 90 consecutive days.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups: 10 males + 10 females
Recovery groups: 5 males + 5 females
Total = 100 (50 males + 50 females)

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels of 50 (G2), 150 (G3) and 300 (G4/G4R) mg/kg/day were selected by the Sponsor.
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and vehicle control recovery groups were handled in a manner similar to the treatment groups except for test item administration.

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS
Clinical Signs and Mortality
Each rat was observed twice daily for morbidity and mortality except during holidays wherein the observation was done once daily as there were no clinical signs observed. Routine observations for checking general clinical signs were performed once a day during treatment (post-dose) period except on day 114, wherein routine observation for checking general clinical signs was not performed. This deviation does not affect the study results.
On the days of scheduled detailed clinical examination (once weekly), clinical signs was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
Detailed Clinical Examination
Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 2 days) during treatment period.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards).
On the days of detailed clinical examination, observation for general clinical signs (first post-dose) was not performed except on Day 1.
Ophthalmological Examination
Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups and at the end of recovery period for recovery groups. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.
Functional Observation Battery Tests (FOB)
The following neurological examination was performed during the 12th week (Day 81 and 82) of treatment period for main groups and towards the end of recovery period (Day 110) for recovery groups.
Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during Removal of Animal from Home Cage and Handling
The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores/ranks.
Open Field Observation
Rat was placed (one at a time) 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 group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and observations were recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- bizarre behaviour
- urination
- defecation
- rearing
- abnormal vocalizations
Functional Tests
Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, ambulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.
Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
- approach response
- touch response
- click response
- tail-pinch response
- pupil response
- aerial righting reflex

Landing Hindlimbs Footsplay
The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the table top 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 suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.
Grip Performance
Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.
Physiological Observations
Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer.
At the end of the functional test, body weight of each rat was measured.
Body Weight
Individual body weights (g) was recorded prior to test item administration on Day 1 and weekly thereafter (± 1 day) for all groups of rats during treatment and recovery period. Fasting body weight was recorded prior to sacrifice for all animals.
Food Consumption
The food consumption was measured at weekly intervals during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.
Oestrous Cycle Evaluation
Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.
Clinical Pathology Investigations
Blood Collection
At the end of treatment period (Day 91) and recovery period (Day 119), all the rats were fasted overnight (water allowed) and approximately 3.0 mL of blood was collected (as indicated in the table below) under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture:
Parameter Approximate volume (mL) Anticoagulant
Name Concentration per mL of blood
Haematology 0.7 K2 EDTA 1.6 mg
Clinical chemistry 1.8 Lithium heparin 10 Unit
Coagulation 0.5 Trisodium citrate 3.2 mg
After analysis and data review by the analyst, the residual samples were disposed.
Haematology
The following haematological parameters were determined using Advia 2120i Hematology system (Siemens Healthcare Diagnostics Inc., NY, USA).
Sl. No. Parameter Abbreviations Units2
1 Red Blood Corpuscles RBC 1012/L
2 Haemoglobin HGB g/L
3 Haematocrit HCT L/L
4 Mean Corpuscular Volume MCV fL
5 Mean Corpuscular Haemoglobin MCH pg
6 Mean Corpuscular Haemoglobin Concentration MCHC g/L
7 Reticulocytes count Retic 1012/L
8 White Blood Corpuscles WBC 109/L
9 Differential leukocyte count1 DLC 109/L
10 Platelets PLT 109/L
1: Differential Leukocyte parameters and their respective abbreviations are: Neutrophils (Neut), Lymphocytes (Lymp), Monocytes (Mono), Eosinophils (Eosi) and Basophils (Baso).
2: Expansion of units are as follows: L:Litre, g: Gram, pg: Picogram, fL:Femtolitre,
Additionally, blood smears were prepared from the haematology (K2EDTA tube) samples and stained with Giemsa stain. As the study findings did not warrant blood smear evaluation, all the slides were discarded.
Coagulation
Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg), at 15°C for 10 minutes for separation of plasma and analysed for the following parameters in plasma sample using STart Max coagulation analyzer (Diagnostica stago, 92600 Asnieres, France):
Sl. No. Parameter Abbreviations Units
1 Prothrombin Time PT Sec
2 Activated Partial Thromboplastin Time APTT Sec
Clinical Chemistry
Plasma was separated after centrifugation of the whole blood samples at 4ºC, 5000 rpm for 5 minutes and analysed using Dimension RxL MaX Clinical Chemistry System (Dade Behring Inc. Newark, DE 19714, USA) for the following parameters:
Sl. No. Parameter Abbreviations Units
1 Alanine Aminotransferase ALT U/L
2 Alkaline Phosphatase Alp U/L
3 Albumin ALB g/L
4 Albumin/Globulin ratio (calculated value) A/G -
5 Aspartate Aminotransferase AST U/L
6 Blood Urea Nitrogen BUN mmol/L
7 Chloride Cl mEq/L
8 Creatinine Creat µmol/L
9 Calcium Ca mmol/L
10 Glucose Glu mmol/L
11 Globulin (calculated value) GLOB g/L
12 HDL Cholesterol HDL.Chol mmol/L
13 Inorganic Phosphorous Pi mmol/L
14 LDL Cholesterol2 LDL.Chol mmol/L
15 Potassium K mEq/L
16 Sodium Na mEq/L
17 Total Cholesterol T. Chol mmol/L
18 Total Plasma Protein T. Pro g/L
19 Triglycerides Trig mmol/L
20 Total Bilirubin T. Bil µmol/L
21 Direct Bilirubin1 D.Bil µmol/L
22 Indirect Bilirubin (calculated)1 Ind.Bil µmol/L
1: Note: Direct and indirect bilirubin were determined when total bilirubin values are
>10 µmol/L
2: LDL Cholesterol was calculated using total Cholesterol, Triglyceride and HDL cholesterol values.
Hormone Analysis
At the end of the treatment and recovery periods, blood was collected from all rats along with blood collection for clinical pathology investigation for the determination of total T3, T4 and TSH hormones.
Blood samples were collected in plain labelled tubes and kept on bench top for 20 min before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4° C. The serum samples were placed in labelled plastic tubes and was stored at ~ -70 °C until they are analysed. The left-over samples from hormone analysis were discarded on the day of data review at the discretion of Study Pathologist.
8.10 Thyroid Profile Hormones:
The following thyroid hormones were estimated by Enzyme Linked Immuno Sorbent Assay (ELISA) method for the samples as stated above using BIO-RAD microplate washer and BIO-RAD model 680 readers.
Sl. No. Parameters Abbreviations Units c
1 Rodent Thyroid Stimulating Hormone TSH ng/mL
2 Rodent Thyroxine T4 ng/mL
3 Rodent Triiodothyronine T3 ng/mL
c: expansion of unit: ng/mL: nano grams/ per milli litre
The kits manufactured by Endocrine Technologies Inc., USA was used for the assay.
Urinalysis
Urine was collected from all rats at the end of the treatment period for main groups and at the end of the recovery period for recovery groups in urine collection tubes.
Each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis.
Urine was analyzed for:
Sl. No. Parameters
1 Specific gravity1
2 Nitrite2
3 pH2
4 Proteins2
5 Glucose2
6 Ketone bodies2
7 Urobilinogen2
8 Bilirubin2
9 Appearance (colour and clarity)3
10 Volume (approximate)3
1: Measured using Refractometer (PAL-10S, Atago Co., Ltd., Japan).
2: Analyzed using Multistix 10 SG strips with Clinitek status analyzer and (Bayer Healthcare LLC, UK).
3: Recorded manually.
Urine was also subjected to microscopic examination as provided below:

Sl. No. Parameters
1 Crystals
2 Epithelial Cells
3 Erythrocytes
4 Leukocytes
5 Casts

Sacrifice and pathology:

Anatomic Pathology
Necropsy
All the rats in the study were subjected to detailed necropsy (examination of external surfaces of the body, all orifices, cranial, thoracic and abdominal cavities and their contents) and findings were recorded. All the rats in the study were subjected to necropsy after overnight fasting (water allowed) at the end of treatment period (Day 91) for main group rats and at the end of recovery period (Day 119) for recovery group rats. Terminal body weights were recorded for all the animals immediately prior to sacrifice. Rats to be sacrificed at term were euthanized by exsanguination under isoflurane anesthesia.
Tissue Collection, Weighing and Preservation
On completion of gross pathology examination, the tissues and organs noted below were collected and weighed from all rats as appropriate. The organ weight ratios (organ to body weight and organ to brain weight) as percentage of fasting body weight and brain weight were determined. The paired organs were weighed together and the combined weights were presented. The tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes and eyes:
Sl. No. Tissues/organ Organ Weighing Collection
and Preservation Microscopic Examination
1. Aorta X X
2. Bone marrow smear1 X X
3. Brain including medulla/pons, cerebellum and cerebrum X X X
4. Cecum X X
5. Cervix X X
6. Colon X X
7. Duodenum X X
8. Epididymides X X X
9. Esophagus X X
10. Eyes 2 X X
11. Femoral muscle
(Skeletal Muscle) X X
12. Femur Bone with joint3 X X
13. Glands, adrenal X X X
14. Glands, salivary X X
15. Gross lesions/masses/nodules X X
16. Gut associated lymphoid tissue X X
17. Heart X X X
18. Ileum X X
19. Jejunum X X
20. Kidneys X X X
21. Larynx X X
22. Liver X X X
23. Lungs (with bronchi and
bronchioles)4 X X
24. Lymph nodes, mandibular X X
25. Lymph nodes, mesenteric X X
26. Mammary gland X X
27. Nerves, sciatic X X
28. Nerve, optic X X
29. Ovaries X X X
30. Oviducts X X
31. Pancreas X X
32. Pharynx X X
33. Pituitary8 X X X
34. Prostate5 X X X
35. Rectum X X
36. Seminal vesicles and coagulating glands5 X X X
37. Skin X X
38. Spinal cord (cervical, thoracic and lumbar) X X
39. Spleen X X X
40. Sternum with marrow3 X X
41. Stomach X X
42. Testes6 X X X
43. Thymus X X X
44. Thyroid and Parathyroid8 X X X
45. Trachea X X
46. Tongue X X
47. Urinary bladder X X
48. Uterus 7 X X X
49. Vagina X X
1 : Collected from femur marrow and stained with Giemsa stain
2 : Collected with optic nerve and preserved in Davidson’s fluid.
3 : Decalcified prior to sectioning
4 : Lungs were inflated with 10%NBF before immersion in the fixative.
5 : Prostate and Seminal vesicles with coagulating glands were weighed as a whole; subsequently prostate was separated and weighed. The derived weight was presented for the seminal vesicles and coagulating glands.
6 : Collected in modified Davidson’s fluid.
7 : Weighed with cervix
8 : Weighed after formalin fixation
X : Activity carried out.


Histopathology
Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4). Histopathological examination of the testes also involved qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically. Further, kidneys from female rats were suspected of showing test item-related histopathological changes in the high dose (G4) group and hence the same was also examined in the lower dose groups (G2, G3) and recovery groups (G1R, G4R).

Statistics:

Data captured using Provantis™ for the parameters body weight and organ weights; laboratory Investigations – Haematology, Coagulation and Clinical Chemistry were analyzed using built-in statistical tests.
Derived data like net body weight change, food consumption and organ weight ratios were also analyzed using above mentioned methods.
The statistical analysis of neurological observations (neuromuscular observation/body temperature/body weights) and the hormones, viz. TSH, T4, T3 data were carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. The data were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before performing ANOVA. Comparison of means between treatment and control groups was done using Dunnett’s test when the overall treatment, ‘F’ test found significant.
The data pertaining to males and female rats was evaluated separately.
All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated by the following symbols throughout the report:
*: Significantly higher/lower than the respective control group

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Higher severity/incidences of renal tubular mineral deposition in kidneys was noted at 300 mg/kg dose in females.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

Higher severity/incidences of renal tubular mineral deposition in kidneys was noted at 300 mg/kg dose in females.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, oral gavage administration of Hordaphos CC MS at 50, 150 and 300 mg/kg/day doses to Wistar rats for 90 consecutive days did not cause any toxicological effects on general health status. The body weights and food consumption were not altered by the treatment at 50, 150 and 300 mg/kg/day doses.
Treatment did not show any adverse effects on haematology, coagulation, clinical chemistry, thyroid profile, urinalysis parameters and gross pathology in both male and female rats at all the dose levels tested.
Microscopically, tubular mineralization in kidneys was characterized by deposition of basophilic, granular or lamellar material in the tubules and/or collecting ducts of the renal outer medulla (outer/inner stripe) with no reaction to these deposits at all the dose levels in females. At 300 mg/kg dose, the deposits were largely multifocal (prominent) and/or bilateral in distribution with minimal to moderate severity and were generally considered to be of higher degree as compared to lower dose groups. Whereas in 50 mg/kg and 150 mg/kg group rats, the mineral deposition varied from focal to multifocal (few) showing unilateral/bilateral distribution with minimal severity, and this was in general comparable to spontaneously observed mineralization pattern in the vehicle control females. Tubular mineralization noted at all the doses were not accompanied by tubular dilation, tubular casts, inflammatory response/fibrosis or any evidence of tubular regeneration in the surrounding tissue. Further, it was not associated with metastatic calcification (systemic effect) or any derangements in renal biomarkers. Higher severity/incidences of renal tubular mineral deposition noted at 300 mg/kg dose as compared 50 mg/kg and 150 mg/kg groups was considered to be test item-related adverse effect. The renal effect noted in 50 mg/kg and 150 mg/kg female rats was considered to be incidental. At the end of 28 Day recovery period, the tubular mineralization noted at 300 mg/kg females showed early signs of recovery. No test item-related histological effects were noted in any of the other tissues of rats.
No Observed Adverse Effect Level (NOAEL):
Daily oral (gavage) administration of Hordaphos CC MS to male and female Wistar rats at dose levels of 50, 150 and 300 mg/kg bw/day for 90 consecutive days resulted in:
• Higher severity/incidences of renal tubular mineral deposition in kidneys was noted at 300 mg/kg dose in females. Based on these observations, No Observed Adverse Effect Level (NOAEL) for females is considered to be 150 mg/kg Bwt/day under the test conditions and doses employed.
• As there were no treatment-related adverse effects observed up to the higjhest dose in males, the No Observed Adverse Effect Level (NOAEL) for males is considered to 300 mg/kg bw/day under the test conditions and doses employed

Executive summary:

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test itemHordaphos CC MSin Wistar rats when administered orally by gavage for 90 consecutive daysand to assess the reversibilityof any effects during a subsequent 28days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL).

The test item was weighed and suspended in vehicle,i.e.,Milli-Q^®water and administered to rats at the graduated dose levels of 50, 150 and 300 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4 ) / high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/ vehicle control recovery (G1R) groups received vehicle Milli-Q^®water alone. The dose volume administered was 10 mL/kg body weight. Each main group in the experiment was comprised of 10 male and 10 female rats and recovery groups comprised of 5 male and 5 female rats.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was notperformedat the test facility.The concentration and stability of the test item in the vehicle were established at 1 and 100 mg/mL concentrations under Eurofins Advinus Study No. G16490. Based on the results, the test item was stable and homogenous in the vehicle up to 5 days when stored at room temperature.

During the conduct of this study, the prepared dose formulations and vehicle (Milli-Q^®water) were analyzed for homogeneity and active ingredient (a.i.) concentration on test Day 1 and during 2^nd(Day 33) and 3^rd(Day 63) month of the treatment.The results indicated that the overall mean concentration ofHordaphos CC MSin the tested formulation was found to be within± 15% of the claimed concentration and the relative standard deviation(%RSD) was less than 10%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smear was examined in the female rats and the stage ofoestrous cycle was recordedprior to necropsy.

All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weights and brain weight. Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4). Histopathological examination of the testes also involved qualitative assessment of stages of spermatogenesis.In addition, all gross lesions from all the animals were examined microscopically. Further, kidneys from female rats were suspected of showing test item-related histopathological changes in the high dose (G4) group and hence the same was also examined in the lower dose groups (G2, G3) and recovery groups (G1R, G4R).

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

Clinical Signs and Mortality:No Clinical signs or mortalities occurred at any of the doses tested.

Ophthalmological Examination:Ophthalmological examination did not reveal any ocular abnormalities.

Neurological Findings:No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

Body Weights:Treatment did not affect body weight at all the tested doses in either sex.

Food Consumption:Treatment did not affect food consumption at all the tested doses in either sex.

Haematology, Coagulation, Clinical Chemistry and urine parameters:There were no test item related alterations observed at any of the tested dose levels in either sex.

Thyroid Hormone Profile:Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.

Terminal Fasting Body Weights and Organ Weights:No significant changes in terminal fasting body weights and organ weights attributed to test item were observedat any of the tested dose levels in either sex.

Gross pathology:There were no test item-related gross pathological changes observed in both sexes

Histopathology:Microscopically, tubular mineralization in kidneys was characterized by deposition of basophilic, granular or lamellar material in the tubules and/or collecting ducts of the renal outer medulla (outer/inner stripe) with no reaction to these deposits at all the dose levels in females. At 300 mg/kg dose, the deposits were largely multifocal (prominent) and/or bilateral in distribution with minimal to moderate severity and were generally considered to be of higher degree as compared to lower dose groups. Whereas in 50 mg/kg and 150 mg/kg group rats, the mineral deposition varied from focal to multifocal (few) showing unilateral/bilateral distribution with minimal severity, and this was in general comparable to spontaneously observed mineralization pattern in the vehicle control females. Tubular mineralization noted at all the doses were not accompanied by tubular dilation, tubular casts, inflammatory response/fibrosis or any evidence of tubular regeneration in the surrounding tissue. Further, it was not associated with metastatic calcification (systemic effect) or any derangements in renal biomarkers. Higher severity/incidences of renal tubular mineral deposition noted at 300 mg/kg dose as compared 50 mg/kg and 150 mg/kg groups was considered to be test item-related adverse effect. The renal effect noted in 50 mg/kg and 150 mg/kg female rats was considered to be incidental. At the end of 28 Day recovery period, the tubular mineralization noted at 300 mg/kg females showed early signs of recovery. No test item-related histological effects were noted in any of the other tissues of rats.

No Observed Adverse Effect Level (NOAEL):

Daily oral (gavage) administration ofHordaphos CC MSto male and female Wistar rats at dose levels of 50, 150 and 300 mg/kg bw/day for 90 consecutive days resulted in:

Higher severity/incidences of renal tubular mineral deposition in kidneys was noted at 300 mg/kg dosein females. Based on these observations, No Observed Adverse Effect Level (NOAEL) for females is considered to be 150 mg/kg Bwt/day under the test conditions and doses employed.