N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide-2-yl)ethane-1,2-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 15, 2016 to September 21, 2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals, No. 408, 21 th September 1998, "Repeated dose 90-day oral toxicity study in rodents"

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Official Journal of the European Union, 31.5.2008, B.26., "Sub-chronic oral toxicity test repeated dose 90-day oral toxicity study in rodents"

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Test Guidelines

Version / remarks:

"90-day Repeated Dose Toxicity Study in Mammalian Species" prescribed in "Concerning Testing Methods Relating to the New Chemical Substances" (March 31, 2011) [Notification 0331 No. 7 of the Pharmaceutical and Food Safety Bureau, Japan's MHLW, No. 5 (March 29, 2011) of the Manufacturing Industries Bureau, Japan's METI & No. 110331009 of the Environmental Health Department, Japan's MOE revised on April 2, 2012: Notification 0402 No. 1 of the Pharmaceutical and Food Safety Bureau, MHLW, No. 2 (March 24, 2012) of the Manufacturing Industries Bureau, METI & No. 120402001 of the Environmental Health Department, MOE].

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one

EC Number:

835-272-7

Cas Number:

256374-76-2

Molecular formula:

C12H26N2O6P2

IUPAC Name:

2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one

Test material form:

solid: particulate/powder

Details on test material:

Chemical name: Reaction products of ethane-1,2-diamine, phosphmyl=trichloride and 2,2-dimethylpropane-1,3-diol which malces N,N-bis(5,5-dimethyl-1,3,2-dioxaphosphinane=2-oxide-2-yl)ethane-1,2-diamine as a main component
Another name: DAIGUARD-850
CAS number: 256374-76-2 (Main component)

Chemical structure etc.
Molecular weight: 356.29 (Main component)

Test sample
Purity of test item: 100%
Lot number: SK-270901

Physicochemical property
Vapor pressure: 11.5 Pa (20°C)
Water solubility: 0.613 ± 0.007 g/L
Partition coefficient: logPow< 1.0
Melting point: 258.5 ± 0.12 °C
Appearance: White powder
Storage conditions: The test sample was stored in a dark storage place at room temperature.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Crl:CD(SD) rats (SPF) were obtained from Charles River Japan Hino Breeding Center. This strain is established as a laboratory animal and widely used in general toxicity studies. In addition we have historical data of this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD(SD) rats (SPF) were obtained from Charles River Japan Hino Breeding Center.
Forty four males and forty four females were obtained at 4 weeks old. The animals were acclimatized and quarantined for 6 days under group housing of four or five animals per cage. No abnormal changes were noted in any animals during the quarantine or acclimation periods. On 6 days after the receipt the animals were allocated to groups using body weight-stratified randomization and housed individually after group allocation, and the body weights of each animal were in the range of ±20% of the mean body weights in each sex. The animals not allocated were excluded from the study.
Clinical conditions and excretions were observed more than once a day from the receipt to the start of dosing.
The animals were identified with a marker on the tail before group allocation, and ear-tags for after group allocation. Cages were identified by individual labels. Racks were identified by indicating of the study number.
At the onset of treatment the animals were 5 weeks old with body weight ranges of 135.6-159.1 g and 118.5-139.9 g, respectively for males and females.

HOUSING CONDITIONS
The barrier-system animal rooms (quarantine room no. 1 and animal room no. 7) were maintained at a stable temperature of21-25°C and relative humidity of 40-70% with 10-15 air changes per hour and artificial light-dark cycle of 12-12 hours (light on: 7:00 and light off: 19:00).
The animals were housed in hanging stainless steel cages with wire-mesh floor of 260 Wx380 Dx 180 Hmm. Undertrays were changed at the end of the quarantine period and at group allocation, and twice a week after group allocation and before carrying animals from the animal room to the autopsy room. Feeders, cages and racks were changed at group allocation and once a month. These caring equipment were autoclaved prior to use.
The animals had free access to a pelleted diet (MF, lot no. 151002, 151124, 151213 and 160107, Oriental Yeast) after autoclave sterilization. Chlorinated water was maintained at 3-5 ppm of chloric level prepared by adding sodium hypochlorite (Purelox) to tap water and given by an automatic watering system.
Information of the contaminants in the diets was obtained from supplier and confirmed to meet the requirements of CERI Rita which referred to the "Toxic Substances Control Act of US-EPA (1979)".
Contaminants in drinking water were analyzed twice a year according to a water regulation of the "Ordinance on drinking water quality standards" [Ordinance No. 101 of MHLW]. Analytical data of the contaminants in water were in the stated ranges of CERI Hita.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

All animals were repeatedly administered daily by gavage for 90 days according to the test methods between 8:11 and 11:55. A syringe (Terumo) connected to a Nelaton catheter (Terumo) was used for the administration. Dosing volume was 10 mL/kg based on the latest body weights.

Vehicle:

olive oil

Details on oral exposure:

Preparation and Storage of Formulations
The test item was weighed and ground with a mortar. Olive oil was added to the powder to make 10.0 w/v% formulation. During the 10.0 w/v% formulation was stirred with a magnetic stirrer a part of the formulation was taken and diluted with the olive oil to prepare the 1.00 and 3.00 w/v% formulations.
The formulations and vehicle were subdivided into plastic containers and stored at a cold place (tolerance temperature 1-10°C). On each dosing day the formulations and vehicle were taken out from the storage place and administered to the animals. The formulations were used within 13 days after preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and Stability Analyses
Homogeneity and stability for 13 days at cold place of the 10.0 and 0.100 w/v% formulations were confirmed in "Validation test for stability of SH-0850 and for unifonnity, stability and concentration of the test substance solution" (study number XlS-1025) in CERJ Hita.

Concentration Analysis
Concentrations of the 10.0, 3.00 and 1.00 w/v% formulations were analyzed by high performance liquid chromatography (HPLC) immediately after the first preparation. Analytical method was decided according to the result of the validation of analytical method (non-GLP, study number Xl 8- 1025).

Pre-treatment
The samples were collected from the formulations (middle layer, n=l) and exactly diluted as below.

10.0 w/v% Formulation
The formulation of0.05 mL and vehicle of 4.95 mL were added on a filter. After the filter was washed with about 5 mL of hexane and filtrated five times, the residue on the filter was diluted with methanol/purified water = 1/1 (v/v) to about 30 mL using ultrasonic irradiation for 2 minutes. This solution was brought to a volume of 50 mL with methanol/purified water= 1/1 (v/v), and served as an analytical sample (dilution rate 1000).

3.00 w/v% Formulation
The formulation of 0.2 mL and vehicle of 4.8 mL were added on a filter. After the filter was washed with about 5 mL of hexane and filtrated five times, the residue on the filter was diluted with methanol/purified water = 1/1 (v/v) to about 30 mL using ultrasonic irradiation for 2 minutes. This solution was brought to a volume of 50 mL with methanol/purified water= 1/1 (v/v), and served as an analytical sample (dilution rate 250).

1.00 w/v% Formulation
The formulation of 0.5 mL and vehicle of 4.5 mL were added on a filter. After the filter was washed with about 5 mL of hexane and filtrated five times, the residue on the filter was diluted with methanol/purified water = 1/1 (v/v) to about 30 mL using ultrasonic irradiation for 2 minutes. This solution was brought to a volume of 50 mL with methanol/purified water = 1/1 (v/v), and served as an analytical sample ( dilution rate 100).

Preparation of standard solution
The test item of 0.10025 g was ~eighed, filled up to the volume of 50 mL with methanol/purified water= 1/1 (v/v), and dissolved by ultrasonic irradiation for 1 minute to prepare 2005.0 μg/mL standard stock solution. Accurate 1 mL of the standard stock solution was filled up to 20 mL with methanol/purified water= 1/1 (v/v) to make 100 μg/mL standard solution.

Analytical conditions
Instruments (HPLC 14)
Pump A andB: L-2130, Hitachi High-Technologies
Auto sampler: L-2200, Hitachi High-Technologies
RI detector: L-2490, Hitachi High-Technologies
Column oven: L-2350, Hitachi High-Technologies
Data processor: EZChrom Elite, Hitachi High-Technologies

Conditions
Column: L-column ODS (4.6 mm I.D. x 150 mm), CERI
Column oven temperature: 40°c
Mobile phase: Methanol/purified water= 1/1 (v/v)
Flow rate: 1 mL/minute
Polarity: +
Injection volume: 10 μL
Auto sampler temperature: 15°c
Auto sampler rinse solution: Methanol/purified water= 1/1 (v/v)

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate dose group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

20 animals/dose (10 male/10 female)

Control animals:

yes, concurrent vehicle

Details on study design:

No abnormal changes were noted in "Twenty-eight day repeated oral dose toxicity study of SH-0850 in the rat" (study number Bll-1025) performed at dose levels of 0, 100, 300 and 1000 mg/kg/day. Therefore the same dose levels were employed in this study.

Positive control:

Not specified

Examinations

Observations and examinations performed and frequency:

GENERAL CLINICAL OBSERVATIONS
Clinical observations including mortality were made in all animals twice a day, i.e., before and just after dosing.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed in all animals once before the dosing period.
Thereafter the animals were observed once a week during the dosing period on a blind test basis.
The blind test was carried out with the random numbers and observation labels without identifying the dose levels.
Observations at removal from cage: Animal reactions such as excitement from external stimuli (holding animals or bringing a hand close to animals to hold) were observed and assessed by scoring
method for ease of removal and vocalization.
Handling observations: Muscle tone, subnormal temperature, hair appearance (piloerection, staining hair and unkempt hair), skin and mucous col or (paleness, reddening and cyanosis), eyes (lacrimation, exophthalmos and pupillary size), salivation and secretion
Observation in arena: Animals were placed in a standard arena (on an observation platform of 90 cm x 60 cm) and observed for posture, motor activity level, respiration, gait characteristics, lid closure, tremor, twitch, convulsion, stereotypical behavior and abnormal behaviour for one minute or more (within five minutes). Frequencies of defecation (number of feces) and urination (number of pools) were recorded for one minute.

BODY WEIGHTS
All animals were weighted with an electric balance (SARTORIUS) on days 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84 and 90, and on the necropsy day.

FOOD CONSUMPTION
Food weights were measured with an electric balance (SARTORIUS) on the following days for all animals: feeding weights on day 1; remainder and feeding weights on days 3, 7, 14, 21, 28, 35, 42, 56, 63, 70, 77 and 84; remainder weights on day 90. Mean food consumption per day was calculated from their feeding and remainder weights.

OPHTHALMOLOGICAL EXAMINATIONS
Ophthalmological examinations were performed for all dose groups before dosing period, and the control and 1000 mg/kg groups in the last dosing week. Anterior eye part, optic media and ocular fundus were examined with slit lamp (SL-5, Kowa) and fundus camera (RC-2, Kowa) after a mydriatic agent of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P, Santen Pharmaceutical) was applied to the eyes.

CLINICAL LABORATORY EXAMINATIONS
Blood Collection and Preparation
Blood was collected from the abdominal aorta under isoflurane anesthesia on the next days of the last dosing after overnight fasting (16 to 20 hours). Blood samples were prepared as follows.
Whole blood: collected in a blood collecting bottle (SB-41, lot no. G5026, Sysmex) with EDTA-2K.
Plasma: collected in a test tube with 100 μL of 3.2 w/v % sodium citrate aqueous solution (lot number CTR6101, Wako Pure Chemical) and centrifuged at 3000 r.p.m. for 10 minutes.
Serum: collected in a test tube and centrifuged at 3000 r.p.m. for 10 minutes.

Hematology
Whole blood or plasma samples of all animals were examined for the following parameters.
Red blood cell count (RBC); Hemoglobin cone. (Hb); Hematocrit value (Ht); Mean corpuscular volume (MCV); Mean corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin conc. (MCHC); Platelet count (Platelet); Reticulocyte count ratio (Reticulo); White blood cell count (WBC); Differentiation of leukocytes [neutrophils (Neutro), lymphocytes (Lymph), eosinophils (Eosino), basophils (Baso), monocytes (Mono)]; Prothrombin time (PT); Activated partial thromboplastin time Plasma(APTT)

Blood Chemistry
Serum samples of all animals were used to determine the following parameters.
Aspartate aminotransferase (AST); Alanine aminotransferase (ALT); Alkaline phosphatase (ALP); r-Giutamyl transpeptidase (y-GTP); Total cholesterol (T-Cho); Triglyceride (TG); Blood urea nitrogen (BUN); Creatinine; Total protein (T-Protein); Albumin; A/G ratio; Glucose; Total bilirubin (T-Bil); Total bile acids (TBA); Inorganic phosphorus (IP); Calcium (Ca); Sodium (Na); Potassium (K); Chloride (Cl)

Sacrifice and pathology:

Gross Necropsy
All animals were subjected to a detailed gross necropsy after blood collection and bleeding from the ventral aorta on the next day of the last dosing. External surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and their contents were observed.

Tissue Collecting and Organ Weight Measurements
The organs and tissues were removed from all animals. Trachea, lungs and urinary bladder were inflated with 10% neutralized buffered formalin before removal. Stomach and intestines were filled and fixed with 10% neutralized buffered formalin and the contents were washed away with water. Parathymic lymph nodes were collected in one female of the 1000 mg/kg group as a macroscopic lesion.
Liver, heart, kidneys, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, brain, spleen, thymus, and adrenals were measured using an electric balance (SARTORIUS). Kidneys, testes, epididymides, ovaries and adrenals were weighed right and left together. Uterus was weighed with the contents. Relative organ weights were calculated based on the body weights measured on the necropsy day.
The testes were fixed with modified Davidson's fixative. The other organs and tissues were preserved in 10% neutralized buffered formalin.
Respiratory system: Trachea, lungs
Salivary glands (submandibular and sublingual glands), esophagus,
Digestive system: stomach, intestines ( duodenum to rectum, including Peyer' s patches), pancreas, liver
Cardiovascular system: Heart, aorta
Urinary system: Kidneys, urinary bladder
Reproductive system: Testes, epididymides, prostate, seminal vesicles, ovaries, uterus, vagina
Nervous system: Brain (including cerebrum, cerebellum and pons), spinal cord (cervical, thoracic and lumber regions), sciatic nerve
Hematopoietic system: Bone marrow (femur), axillar lymph nodes, mesenteric lymph nodes, spleen, thymus
Endocrine system: Pituitary gland, thyroid (including parathyroid), adrenals
Sense organ: Eye balls
Skin and adnex Skin: mammary glands

Histopathological Examinations
Light microscopic examinations were performed for the organs and tissues after embedding in paraffin, sectioning and hematoxylin and eosin (HE) staining. Decalcification was done for the bone marrow (femur) with 10% formic acid formalin before trimming. Macroscopic lesions were examined: testes of one male in the 100 mg/kg group; parathymic lymph nodes in one female of the 1000 mg/kg group.
Manufacturing of the histopathological specimen, from thin sectioning to HE staining and mounting, was performed at Sapporo General Pathology Laboratory.
Trachea; Lungs; Salivary glands; Esophagus; Stomach; Duodenum-rectum; Pancreas; Liver; Heart; Aorta; Kidneys; Urinary bladder; Testes; Epididvmides; Prostate; Seminal vesicle; Ovaries; Uterus; Vagina; Cerebrum; Cerebellum; Pons/medulla; Spinal cord; Sciatic nerve; Bone marrow; Axillar lymph nodes; Mesenteric lymph nodes; Spleen; Thymus; Pituitary gland; Thyroid; Parathyroid; Adrenals; Eye balls; Skin; Mammary glands

Statistics:

Data regarding body weights, food consumption, parameters of hematology and blood chemistry, organ weights and body weights on the necropsy day were analyzed by the Bartlett's test for homogeneity of variances. When the variances were homogeneous at a significance level of 5% in this analysis, the Dunnet's test was performed. When the variances were not homogeneous, the nonparametric Dunnett's test was performed. The frequencies of defecation and urination were analyzed by the nonparametric Dunnett's test.
The analyses were performed by a two-tail test, and results were regarded to be significant when the calculated P-values were 1 or 5% compared to the relevant control values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

In males, defecation was decreased in the 100 mg/kg group in week 4 and in the 300 mg/kg group
in week 5 without dose-relationships. No abnormal changes were observed in the 1000 mg/kg group.
In females, excessive responses were noted during the animals were taken out from their home cages in one each of the control and 100 mg/kg groups in week 1. This change showed no dose-relationship.
No abnormal signs were noted in the 300 or 1000 mg/kg groups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males or females, body weights were not affected in any dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, food consumption was increased in the 1000 mg/kg group on day 90. No abnormal food intakes were noted in the 100 or 300 mg/kg groups.
No abnormal food intakes were noted during the dosing period in females of any dose levels.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No abnormal changes were observed in males or females of any dose groups before dosing period or last dosing week.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, although activated partial thromboplastin time (APTT) was shortened in the 1000 mg/kg group, this change was within our historical control range. No hematological changes were noted in the 100 or 300 mg/kg groups.
In females, white blood cell count (WBC) was increased in the 100 mg/kg group. Eosinophils were increased in the 300 mg/kg group. These changes showed no dose-relationships. Although monocytes were increased in the 100 and 1000 mg/kg groups, these changes were within our historical control ranges and to be no dose-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, albumin and A/G ratio were decreased in the 100 mg/kg group. r-Glutamyl transpeptidase (r-GTP) was increased in the 300 mg/kg group. These changes were to be no dose-related. No abnormal changes were noted in the 1000 mg/kg group.
In females, sodium was decreased in the 100 mg/kg group, and alanine aminotransferase (ALT) was increased in the 300 mg/kg group without dose-relationships. Abnormal parameters were not noted in the 1000 mg/kg group.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males, relative weights of the testes and seminal vesicles were decreased in the 300 mg/kg group without dose-relationships. Abnormal organ weights were not noted in the 100 or 1000 mg/kg groups.
No abnormal organ weights were noted in females of any dose groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, bilateral small testes were observed in one of the 300 mg/kg group. Blackish region of the mucosa of the glandular stomach was observed in one of the 1000 mg/kg group. Diverticulum in the jejunum was observed in one of the 1000 mg/kg group. No abnormal changes were observed in the 300 mg/kg group.
In females, dark reddish region in the lungs and enlargement of the parathymic lymph nodes were observed in one of the 1000 mg/kg group. No macroscopic changes were observed in the 100 or 300 mg/kg group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In males, only spontaneous lesions were observed: focal myocarditis in three of the control group; lymphocyte infiltration in the prostate in three of the control group; aberrant craniopharyngeal tissue in the pituitary gland in one of the control group; diffuse hyperplasia of the Leydig cells and bilateral diffuse atrophy of the seminiferous tubules of the testes in one of the 100 mg/kg group; squamous epithelial cyst in the forestomach in one of the 1000 mg/kg group; focal necrosis of the fundic mucosa of the glandular stomach in one of the 1000 mg/kg group; diverticulum in the jejunum in one of the 1000 mg/kg group; focal myocarditis in two of the 1000 mg/kg group; lymphocyte infiltration in the prostate in two of the 1000 mg/kg group. The examinations were not performed in the 300 mg/kg group.
In females, following changes were spontaneous lesions: focal inflammation in the lungs in one of the control group; mineralization in the corticomedullary junction of the kidneys in four of the control group; ultimobranchial rest in the thyroid in one of the control group; mineralization in the corticomedullary junction of the kidneys in two of the 1000 mg/kg group; hypertrophy of the paracortex of the parathymic lymph nodes in one of the 1000 mg/kg group; Rathke's pouch remenant in the pituitary gland in one of the 1000 mg/kg group. Any animals in the 100 or 300 mg/kg groups were not histopathologaically examined.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Although food consumption was increased in males of the 1000 mg/kg group on day 90, this change did not affect to the body weights and was considered to be no toxicologically significance. In the hematology, statistically significant changes of APTT and monocytes were noted in the 1000 mg/kg group. These changes were considered to be accidental ones since they were within our historical control ranges, and there were no related-changes to them.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects noted

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Figures and Tables showing the results can be found attached under background information.

Applicant's summary and conclusion

Conclusions:

Treatment of the test item for 90 days produced no treatment-related changes in any test parameters. The test item was considered to show no toxicity under the conditions tested.
Although food consumption was increased in males of the 1000 mg/kg group on day 90, this change did not affect to the body weights and was considered to be no toxicologically significance. In the hematology, statistically significant changes of APTT and monocytes were noted in the 1000 mg/kg group. These changes were considered to be accidental ones since they were within our historical control ranges, and there were no related-changes to them.
The No-Observed-Adverse-Effect Level (NOAEL) of DAIGUARD-850 was estimated to be 1000 mg/kg/day under the conditions tested.

Executive summary:

A 90-day repeated-dose oral toxicity study of DAIGUARD-850 was performed to characterize the toxicity of the test item in accordance with Concerning Testing Methods Relating to the New Chemical Substances, Japan, OECD TG408 and EN B.26.

 

Male and female Crl:CD(SD) rats at 5 weeks old were treated orally with the test item mixed with olive oil for 90 days. During the dosing period, general clinical observations, detailed clinical observations, body weights and food consumption measurements, and ophthalmological examinations were performed. On the next day of the last dosing, blood and pathological examinations were performed. The dose levels were set at 0 (olive oil), 100, 300 and 1000 mg/kg/day, and 10 males and 10 females were used for each dose level.

 

The test item did not affect to any test parameters, and the No-Observed-Adverse-Effect Level (NOAEL) of DAIGUARD-850 was considered to be 1000 mg/kg bw/day, the highest dose level used in the study.