Reaction mass of isopropyl dihydrogen phosphate and phosphoric acid and propan-2-ol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 July 2015 to 23 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of diisopropyl hydrogen phosphate and {[hydroxy(propan-2-yloxy)phosphoryl]oxy}(propan-2-yloxy)phosphinic acid and isopropyl dihydrogen phosphate and methyl dihydrogen phosphate and phosphoric acid

EC Number:

908-995-1

Molecular formula:

can not be determined for reaction masses

IUPAC Name:

Reaction mass of diisopropyl hydrogen phosphate and {[hydroxy(propan-2-yloxy)phosphoryl]oxy}(propan-2-yloxy)phosphinic acid and isopropyl dihydrogen phosphate and methyl dihydrogen phosphate and phosphoric acid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Test item formulations were prepared daily before administration. The required quantity of test item was weighed into a clean glass beaker and there by adding little volume of vehicle into the beaker, mixed well with glass rod and transferred into the measuring cylinder. Again a small quantity of vehicle was added, mixed well with glass rod to rinse the beaker and transferred into the measuring cylinder. The rinsing procedure was repeated to
ensure complete transfer of the test item formulation into a measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get a desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.

VEHICLE

- Justification for use and choice of vehicle (if other than water): The test item was clearly miscible in distilled water as evidenced by the inhouse miscibility test. Hence, distilled water was selected for the test item formulation preparation.
- Concentration in vehicle: 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.
- Amount of vehicle (if gavage): 10 mL/kg body weight.
- Lot/batch no. (if required): Distilled Water Batch No.: 5108, Expiry Date: 06/2016; Batch No.: 5089, Expiry Date: 07/2016; Batch No.: 5103, Expiry Date: 08/2016; Batch No.: 5193., Expiry Date: 08/2016.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis for dose concentration verification of Hordaphos CC MIS was done by Analytical Chemistry of Bioneeds India Private Limited. The analysis was done as per methods detailed in the Study Plan No. BIO-ANM 116 Sampling and analysis of formulations were performed on first day of treatment and during week 5 of the treatment. The samples were collected in duplicates from each concentration at volume of 5 mL (5 mLx2).
The collected samples were transferred to Analytical Chemistry of Bioneeds India Private Limited for dose concentration analysis. One set of aliquot of each formulation were analyzed. The second aliquots were stored as a back up purpose at room temperature. The second set of samples was discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is equal to or less than 10.0%.

Duration of treatment / exposure:

The test item or vehicle was administered to animals through oral (gavage) route once daily.

The main group males were treated for two weeks premating, during mating and up to the day before sacrifice during postmating period (total of 35 days of treatment).

The main group females were treated for two weeks premating period, during mating, pregnancy (gestation) and up to lactation day 4 after which the pups were sacrificed on lactation Day 4 and females (dams) were sacrificed on lactation day 5 after overnight fasting (water allowed).

The recovery group animals were treated till first scheduled sacrifice of dam(s) and were observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

The test item/vehicle was administered by oral (gavage) route using intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 10 mL/kg with the concentration of 10, 30 and 100 mg/mL for low, mid and high dose/high dose and recovery groups, respectively. Distilled water was administered to vehicle control/vehicle control recovery group at an equivolume of 10 mL/kg body weight. The actualdose volume for each animal was calculated based on the most recent body weight. The test item formulations were administered as soon as possible after preparation.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Main Group: 24 (12 Males + 12 Females) per dose
Recovery Group: 10 (5 males + 5 females) per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/ high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item Hordaphos CC MIS when administered through oral (gavage) for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1022).

These doses were selected as the test item Hordaphos CC MIS when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (50, 300 and 800 mg/kg body weight).

Positive control:

Not Applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Time schedule: All the animals were subjected to detailed clinical examinations on day 1 before treatment and weekly thereafter during treatment and recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group and recovery group males and females were weighed on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and within 24 hours of parturition (day 1 postpartum) and on day 4 postpartum during lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

OPHTHALMOLOGY:
- Yes, once before treatment for all animals, during end of the dosing period for males (Day 32 - shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice) for vehicle control and high dose main group animals and during last week for recovery group animals (Day 53). The examination was not extended to lower dose groups as there were no treatment related changes noted in high dose group animals.

FOB:
- Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the dosing period for males (Day shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice).
The Neurological/Functional examination was performed for recovery groups during the last week of the recovery period (Day 54).

HAEMATOLOGY:
- Blood samples of both males and females were collected on the day of scheduled terminal sacrifice (for males after completion of 35 days of treatment and for females on lactation day 5).
Analysed parameters: haemoglobin, haematocrit, erythrocyte count, leucocyte count, platelet count, differential leucocytes count, prothrombin time, Activated Partial Thromboplastin Time

CLINICAL CHEMISTRY:
- Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total protein, Albumin, Total bilirubin, Glucose, Total cholesterol, Creatinine, Blood urea nitrogen, Triglycerides, Phosphorous, Calcium, Globulin, Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L)

URINALYSIS:
- Urine was collected from the five randomly selected males of each group during the last week of the treatment period for main groups and during last week of recovery period for recovery groups.
The selected animals were placed in urine collection cages overnight (approximately 12-16 hrs) and not given access to food but water was provided ad libitum during their stay in urine collection cages. The overnight urine volume (mL) collected from these animals was measured.
The volume of urine collected (mL), appearance and the color were recorded by physical evaluation.
Analysed parameters: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Albumin, Leucocytes

Sacrifice and pathology:

SACRIFICE
- Male animals: All animals [after completion of 35 days of treatment.]
- Maternal animals: All animals [on lactation day 5]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY
- Testes, epididymides, liver, kidneys, adrenals, thymus, spleen, brain, heart, ovaries, prostate with seminal vesicles, organs showing macroscoipc lesions, gross lesions, spinal cord, stomach, duodenum, jejunum, ileum with Peyer´s patches, caecum, colon, rectum, thyroid, trachea, lungs, uterus, urinary bladder, mandibular lymph nodes, mesenteric lymph nodes, sciatic nerve, bone marrow

ORGAN WEIGHTS
- Testes, epididymides, liver, kidneys, adrenals, thymus, spleen, brain, heart

Statistics:

The data was subjected to various statistical analyses using SPSS software version 22.

All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aforementioned tests are designated by the superscripts throughout the report as stated below:

* Statistically significant (P<0.05) change than the vehicle control group.
Note: Data of nonpregnant females and females mated but not littered was excluded from statistical analysis.

The statistical analyses were followed to the parameters:
Body weight, Change in body weight, Feed consumption, Copulatory interval, Gestation length, Hematology, Urinalysis, Clinical chemistry, Absolute organ weights, Relative organ weights were analysed by Oneway ANOVA with Dunnett’s posttest for main groups and t-test for recovery groups (Parametric).

FOB ( Body temperature, Defecation, Urination, Rearing, Grip strength, Movements), Mean litter weights, Mean pup weight, Pre implantation loss, Pre natal loss, Post natal loss,Total No. of resorptions/dam, Total No. of early/late resorptions/dam were analysed by Oneway ANOVA with Dunnett’s posttest (Parametric).

Dams with live young born, with early/late resorptions, Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Litter size, Pup weight were analysed by KruskalWallis followed by the MannWhitney test.

Pregnancy rate, No. of live pups, No. of dead pups, No. of early resorptions, No. of late resorptions, No. of litters with/without dead pups, No. of litterswith/without resorptions were analysed by Chisquare test/ Fischer's Exact Test (Cross Tabs)

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

Description (incidence and severity):

There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the doses tested in both the sex.
However, statistical significant decrease in percent change in body weight with respect to day 1 on day 7 in G2 (females) and G4 (females) and statistical significant decrease in percent change in body weight with respect to day 1 on day 7 in G4R (males) was noted when compared with vehicle control group. These changes are considered incidental due to lack of dose dependency and also the mean body weights were comparable.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in haematology at any of the dose tested in both the sex. However, statistical significant increase in percent neutrophils, and statistical significant decrease in percent lymphocytes in G2 (males), statistical significant decrease in total leucocyte count in G4R (females), statistical significant decrease in prothrombin time in G4R (males), statistical significant decrease in absolute lymphocytes, monocytes, eosinophils and basophils in G4R (females) was noted when compared with vehicle control group. These changes were considered incidental as the values were within historical range or due to random biological variation

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in clinical chemistry at any of the dose tested in both the sex.
However, statistical significant decrease in total protein and calcium levels in G3 (females) and statistical significant decrease in alkaline phosphatase and potassium levels in G4R (females) were noted when compared with vehicle control group. These changes were considered incidental as the values were within historical range or due to random biological variation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related changes observed in the absolute and relative organ weights at any of the dose tested in both the sex.
However, statistical significant increase (slight) in relative testes weight in G4 (males) and statistical significant increase in relative brain weight in G2 (females) and G4R (males) were noted when compared with vehicle control group. All the observed changes were considered incidental and not treatment related because of the lack of dose dependency and/or there were no associated macroscopic and microscopic abnormalities noted.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to limit dose

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item Hordaphos CC MIS was found to be 1000 mg/kg body weight when administerd to the main group males for two weeks pre-mating, during mating and up to the day before sacrifice during post- mating period (total of 35 days), the main group females treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day and the recovery group animals till first scheduled sacrifice of dam(s) (i.e. 41 days) by oral (gavage) to Sprague Dawley rats under the experimental conditions and the doses employed.

Executive summary:

The test item, Hordaphos CC MIS, was evaluated for possible adverse effects following repeated oral dosing for relatively limited period of time and to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to main and recovery groups. Each main group (G1, G2, G3and G4) consisted of 12 males and 12 females and recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R groups were administered with vehicle [Distilled water], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered at the dose volume of 10 mL/kg body weight.

The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 35 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 4 after which the pups were sacrificed on lactation Day 4 and females (dams) were sacrificed on lactation day 5 after overnight fasting (water allowed).

The recovery group animals were treated till first scheduled sacrifice of dam(s) and observed for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects.

The test item formulations at the concentrations 10 mg/mL (low dose) and 100 mg/mL (high dose) were stable up to 6 hours at room temperature as per the in-house stability study results (Bioneeds Study No.: BIO-ANM 116). However, freshly prepared test item formulations were administered to the animals.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination was carried out once before treatment for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice)for vehicle control and high dose main group animals andduring last weekfor recovery group animals. Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice). The Neurological/Functional examination was performed for recovery groups during the last week of the recovery period.

The clinical pathology (haematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) and for all recovery group animals. Urinalysis was performed from the five randomly selected males of each group during the last week of the treatment period for main groups and during last week of recovery period for recovery groups.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded. Body weights of male and female pups were recorded separately on lactation days 1 and 4.

Gross pathology and organ weighing were performed on day 36 for main group males, on lactation day 4 for pups, lactation day 5 for dams and at end of recovery period for recovery group males and females. The number of corpora lutea, implantation sites and resorptions for dams were recorded.

Histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure).

The animals did not reveal any clinical signs of toxicity throughout the treatment and recovery period. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis, organ weights (both absolute and relative) were observed in both the sex.

The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights.

The animals (main group and recovery) did not reveal any treatment related gross as well as histopathological changes on examination.