Dialuminium zinc tetraoxide

Endpoint summary

Description of key information

An oral repeated dose 28-Day toxicity study in rodents (OECD 407) is available.

NOAEL (oral, rat) >= 1000 mg/kg bw/day (m/f) for systemic toxicity

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 04, 2016 - April 24, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 3, 2008

Deviations:

yes

Remarks:

No analytic was performed as the test substance is a poor soluble inorganic substance, that is stable under the conditions. The test material was freshly prepared every day.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Co., LTD.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 184.5 to 213.9 g of males and 157.4 to 186.7 g of females
- Fasting period before study: none
- Housing: ≤ 5 animals of same sex were housed in polycarbonate cages (L x W x H: 400mm x 250mm x 200 mm) bedding with corn cob.
- Diet: Irradiated rodent diet (Shanghai Puluteng Bio-technology Co., Ltd, Feed Production Certificate No: Hu Si Zheng (2014) 04001), Lot. No.: M02-F-20160419004 (valid till 18. Oct 2016); ad libitum
- Water: drinking water purified by reverse osmosis, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
The bedding material evaluation for contaminants was performed according to the national standard GB 14924.2-2001. The food evaluation for nutrients was performed by the supplier according to the Chinese national standard GB 14924.3-2010, for contaminants according to GB 14924.2-2001. The total bacteria numbers evaluation of water was performed according to the national standard for drinking water GB 5749-2006 monthly in the facility and the evaluation for contaminations was performed twice a year by the supplier according to GB 5749-2006. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.85 to 24.90, automatically controlled by air conditioner (Honeywell EBI)
- Humidity (%): 46.45 to 71.20
- Photoperiod (hrs dark / hrs light): 12/12 (from 7:30 to 19:30), artificial lighting

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Water for injection

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
The test article was accurately weighed for each dose level, and the vehicle (control article) was added to achieve the prescribed concentrations. The suspsions were stirred with a stirrer. The formulation was stored at room temperature ( 15 - 25 °C) for the use on the day.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): M16040405 and M16061014

Analytical verification of doses or concentrations:

no

Remarks:

No analytic was performed. The test material was freshly prepared every day. During the dosing process, the formulations were continuously stirred on the magnet mixer.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 male/10 female for control and high dose group (1000 mg/kg bw/day)
5 male/5 female for mid and low dose group (100 mg/kg bw/day and 300 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Based on the result of a single dose exposure study (LD50 > 2000 mg/kg bw)
- Post-exposure recovery period in satellite groups: yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality, consumption of food and water

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule of regular clinical observation: All animals were observed each day after dosing for signs of appearance, behavioural changes, mental state, glandular secretion, respiratory status, faecal traits and colour, genitals, mortality and other toxic manifestations.
- Time schedule of detailed clinical observations: Once before dosing, once a week from week 1 to week 4, and once in the last recovery week all animals were observed in home-cage observation, hand-held observation, open-field observation, and stimulation reflex test. The open-field observation of each animal was about 3 minutes.
- Grip strength: Once in the 4th week of treatment and last week of recovery using Grip Strength Meter.


BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed once a week. The last measurement was performed prior to necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: The quantity of food consumed in each cage was measured at weekly intervals throughout the study.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the scheduled day of necropsy
- Anaesthetic used for blood collection: Yes, 60 mg/kg bw pentobarbital sodium, ip
- Animals fasted: Yes, 12 hours prior to blood collection
- How many animals: 5 rats/sex of each treatment group; 5 rats/sex of control group and 1000 mg/kg bw of the recovery groups
- Parameters examined: Red blood cell count (RBC), Hematocrit (HCT), Hemoglobin concentration (HGB), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Reticulocyte count (RET), Reticulocyte count percentage (%RET), White blood cell count (WBC), Differential count of WBC (Neutrophil, Eosinophil, Basophil, Monocyte and Lymphocyte), Platelet count (PLT), Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the scheduled day of necropsy
- Animals fasted: Yes, 12 hours prior to blood collection
- How many animals: 5 rats/sex of each dose groups and 5 rats/sex of control group and 1000 mg/kg bw/day group
- Parameters checked: Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), Creatine kinase (CK), Lactate dehydrogenase (LDH), Total bilirubin (T-Bil), Urea nitrogen (UN) ,Creatinine (Crea), Total protein (TP), Albumin/globulin ratio (A/G ratio), Albumin (ALB), Glucose (GLU), Cholesterol (CHOL), Triglycerides (TG), Sodium (Na+), Calcium (Ca), Potassium (K+), Chloride (Cl-) and Phosphorus (P)

URINALYSIS: Yes
- Time schedule for collection of urine: the last week of dosing and recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Urine volume, Appearance, Glucose (GLU), Bilirubin (BIL), Ketones (KET), Specific gravity (SG), Occult blood (BLO), pH, Total protein (PRO), Urobilinogen (URO), Nitrites (NIT), White blood cell (WBC)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHERS: Sexual cycle
- Time schedule for sampling: on the scheduled day of necropsy
- How many animals: all females
- Parameters checked: Vaginal Smears

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The macroscopic appearance of the tissues of all rats was recorded and samples of the following tissues were preserved for histopathology: adrenals, aorta, brain (medullary, cerebellar and cerebral sections), breast, caecum, colon, duodenum, epididymides, eye, fallopian tube , femur (with joint, bone marrow ), heart, hypophysis, ileum (incl. Peyer's patches), jejunum, kidneys, liver, lymph nodes (mandibular and mesentric), lungs (all lobes, mainstem bronchi), lymph nodes, mesentery lymph node, oesophagus, optic nerve, ovaries, parathyroid gland, pancreas, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle (biceps femoris), skin (groin), spinal cord (neck, chest, waist), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus (with cervix), vagina, other macroscopically abnormal tissue.

Testes/epididymides, eye/optic nerve were fixed in modified Davidson‘s solution. All other tissues were preserved in buffered 10% formalin.

The following ogans from each animal were weighed: brain, spleen, thymus, liver, kidneys, heart, adrenals, testis, epididymides, prostate with seminal vesicle and coagulating gland, uterus, ovary thyroid parathyroid gland


HISTOPATHOLOGY: Yes
Microsccopic examination of prepared slides (from tissues indicated under gross pathology) was carried out for all animals of the control and high-dose group.

Statistics:

Quantitative data such as body weight, food consumption (excluding recovery period data), haematology, clinical chemistry, urine volume, organ weight and ratios, number of rearing, number of faecal particles, number of urination, grip strength were presented as group mean ± standard deviation. Qualitative data (binomial categories data, unordered multi-categories data and ordinal data) such as urinary analysis, home-cage observation, hand-held observation, open-field observation (excluding number of rearing, number of faecal particles, number of urination) and stimulus response observation were presented as observed counts (frequency). When sample size was less than three, raw data of that group were directly presented without statistical comparison.
All pairwise comparison was performed between control group and each treatment group.
Analyses were performed according to sex at two-tailed probability level (α=0.05). PRISTIMA 6.1.1 was used for statistical analysis of body weight, food consumption, haematology, clinical chemistry, organ weight and ratios. The other data were statistically analyzed with Statistical Product and Service Solutions (SPSS 13.0 for Windows).
The results of histopathology were described.

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities were observed in spontaneous activity, general state, skin and hair of all groups of animals in the dosing and recovery period.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in all test substance groups of animals when compared to the control during the dosing and recovery period (p>0.05).

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was statistically significant increased for males (100 mg/kg bw/day group) on Day 7 and for females (1000 mg/kg bw/day) on recovery Day 13. A statistically significant decreased food consumption for males (1000 mg/kg bw/day) on Day 27 (p<0.05) was observed. These changes are considered to be normal test fluctuations and have no toxicological relevance.

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):

No statistically significant differences were observed in all groups of animals when compared to the control group during the dosing and recovery period (p>0.05).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increased glucose (GLU) levels at 300 mg/kg bw/day and 1000 mg/kg bw/day, as well as statistically significant increased phospor (P) levels at 1000 mg/kg bw/day were observed in females at the end of dosing the period when compared to the respective controls. Statistically significant decreased triglyceride (TG) levels were observed in males at 300 mg/kg bw/day at the end of dosing period compared to the control group. A statistically significant increased total bilirubin (T-Bil) level at 1000 mg/kg bw at the end of recovery was observed in males when compared to the control group. However, all these observations are not considered toxicologically relevant for the following reasons: The decrease of TG was not dose-related and the difference was slight. The increase of T-Bil occurred in the recovery period and was within the fluctuation range of animals at the same age in the laboratory (0.37 – 1.8 µmol/L). The increase of GLU and P was slight compared to the control and within the fluctuation range of females at the same age in the laboratory (GLU: 4.59 – 8.32 mmol/L, P: 1.70 – 2.96 mmol//L).

In addition, at the end of the treatment period there was no statistically significant differences between treatment and control groups in ALP, ALT, AST, urea, Crea, CK, LDH, ALB, A/G, CHOL, TP, Ca, Cl-, K+, Na+ in females and males (p> 0.05). At the end of the recovery period, the difference of blood biochemical indices at 1000 mg/kg bw/day group were not statistically significant different compared to the control groups both in females and males (p>0.05).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No statistically significant difference in any of the urinary parameters between treament and control groups was observed at the end of treatment and recovery period (p>0.05).

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in grip strength was observed in females at 1000 mg/kg bw/day in the 4th week of treatment and in males at the end of recovery period (p<0.05). Those observations are not considered toxiclogical relevant as it is an increased in grip strength only. In addition, there was no statistical significant difference from control group observed on grip strength in males at 100, 300 and 1000 mg/kg bw/day group in the 4th week of treatment as well as in females at 100 and 300 mg/kg bw/day. At the end of the recovery period treated female animals did not show a statistically significant difference compared to the controls in grip strength. Since the differences were slight and observed on grasping only, this is considered not treamtent-related.

No statistically significant differences were observed in the hand-held observations, open-field observation in animals of treatment groups compared to the control groups at the end of dosing and recovery period (p>0.05).

No statistically significant difference in any treatment group in stimulus response, i.e. alertness response, abdominal tension, auricle reflex, pinch reflex, reflexive reflex and pupil reflex was observed compared to the control group (p>0.05) on Day 5, 12 19 and 26 and on Day 12 of the recovery period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences were observed in females at 1000 mg/kg bw/day with decreased thymus weight, thymus/body weight ratio and thymus/brain weight ratio compared to the control group at the end of the recovery period (p<0.05). The differences were slight (<= 16.85%) and it appeared in recovery period only. Furthermore, as no statistically significance was observed in the dosing period and no organ abnormalities were observed in the histopathological examinations, the observation was considered to be normal test fluctuations and are considered not toxicologically relevant.
No statistically significant differences of treated animals compared to the control groups were observed in the weight and weight ratio of other organs at the end of the treament period as well as at the end of the recovery period (p>0.05).

Slight increase of uterine weight was observed in individual females. The vaginal smear cycle examination, which was made on the day of necropsy, revealed that the animals with increased weight of the uterus were in the emotional period. Thus, this is the normal phenomenon of the estrus, not treatment related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related gross findings were observed in brain, heart, liver, lung, kidney, spleen, gastrointestinal tract and germinal system at the end of dosing and recovery period.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment related pathological alterations were observed in brain, heart, liver, lung, kidney, spleen, thymus gland, ovary, uterus, testes, epididymides, pancreas, bone marrow, gastrointestinal tract and other organs at the end of dosing and recovery period.
There were no statistically significant abnormalities observed in the treatment groups compared to control groups in the histopathological examinations in ovaries/uterus/vagina with the vaginal smear cycle examination made on the day of necropsy.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related adverse effects observed

Key result

Critical effects observed:

no

Table 1 Effects on Body Weight of Males (g)

Detection time	Control		100 mg/kg bw/day group		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
Pre-dosing	10	201.5 ± 8.7	5	203.9 ± 6.5	5	203.2 ± 8.4	10	201.1 ± 8.2
Day 5	10	249.5 ± 13.4	5	249.0 ± 9.4	5	249.1 ± 10.5	10	247.4 ± 9.6
Day 12	10	303.8 ± 20.1	5	307.7 ± 15.4	5	303.4 ± 15.0	10	305.1 ± 16.5
Day 19	10	348.8 ± 25.7	5	358.8 ± 22.2	5	340.3 ± 21.5	10	350.5 ± 24.3
Day 26	10	392.4 ± 33.6	5	401.7 ± 29.0	5	383.3 ± 18.4	10	395.6 ± 33.3
Recovery day 5	5	405.7 ± 46.4	/	/	/	/	5	425.9 ± 47.3
Recovery day 12	5	429.7 ± 53.7	/	/	/	/	5	450.2 ± 51.4

Notes: * Statistical significance was noted when compared with the control (p≤0.05).

 

Table 2 Effects for D-Prism via Oral Gavage on Body Weight of Females (g)

Detection time	Control		100 mg/kg bw/day group		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
Pre-dosing	10	173.1 ± 7.4	5	172.6 ± 8.9	5	172.3 ± 8.9	10	174.3 ± 6.3
Day 5	10	191.7 ± 7.9	5	191.2 ± 6.3	5	184.3 ± 9.4	10	191.9 ± 4.8
Day 12	10	210.4 ± 15.0	5	213.2 ± 9.5	5	206.1 ± 9.8	10	209.8 ± 11.0
Day 19	10	225.6 ± 18.7	5	228.7 ± 11.9	5	220.8 ± 13.2	10	221.8 ± 12.3
Day 26	10	231.4 ± 20.9	5	243.6 ± 18.3	5	235.9 ± 16.6	10	232.8 ± 18.6
Recovery day 5	5	235.6 ± 27.3	/	/	/	/	5	237.3 ± 18.8
Recovery day 12	5	244.0 ± 27.4	/	/	/	/	5	245.6 ± 13.4

 

Table 3 Effects on Hematology of Males

Parameters Detection time	Control		100 mg/kg bw/day group		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
WBC (10^9/L)
At necropsy of the dosing period	5	5.36 ± 0.89	5	6.49 ± 1.27	5	6.05 ± 1.94	5	6.34 ± 2.59
At necropsy of the recovery period	5	5.36 ± 1.20	/	/	/	/	5	5.50 ± 1.56
NEU% (%)
At necropsy of the dosing period	5	14.8 ± 3.6	5	12.4 ± 3.5	5	14.2 ± 4.2	5	14.8 ± 2.7
At necropsy of the recovery period	5	14.3 ± 3.3	/	/	/	/	5	18.1 ± 4.7
LYM% (%)
At necropsy of the dosing period	5	81.5 ± 3.6	5	83.7 ± 4.3	5	81.8 ± 3.8	5	81.7 ± 2.7
At necropsy of the recovery period	5	81.9 ± 3.4	/	/	/	/	5	78.1 ± 5.2
MONO% (%)
At necropsy of the dosing period	5	1.6 ± 0.6	5	1.7 ± 1.0	5	1.4 ± 0.1	5	1.3 ± 0.3
At necropsy of the recovery period	5	1.8 ± 0.3	/	/	/	/	5	1.7 ± 0.6
EOS% (%)
At necropsy of the dosing period	5	0.82 ± 0.08	5	0.94 ± 0.21	5	1.04 ± 0.09	5	1.02 ± 0.47
At necropsy of the recovery period	5	0.96 ± 0.23	/	/	/	/	5	1.06 ± 0.24
BASO% (%)
At necropsy of the dosing period	5	0.22 ± 0.08	5	0.32 ± 0.11	5	0.28 ± 0.08	5	0.28 ± 0.08
At necropsy of the recovery period	5	0.14 ± 0.05	/	/	/	/	5	0.20 ± 0.07
NEU (10^9/L)
At necropsy of the dosing period	5	0.80 ± 0.27	5	0.84 ± 0.38	5	0.87 ± 0.38	5	0.88 ± 0.13
At necropsy of the recovery period	5	0.75 ± 0.18	/	/	/	/	5	1.01 ± 0.40
LYM (10^9/L)
At necropsy of the dosing period	5	4.36 ± 0.72	5	5.39 ± 0.77	5	4.94 ± 1.60	5	5.24 ± 2.38
At necropsy of the recovery period	5	4.41 ± 1.09	/	/	/	/	5	4.28 ± 1.18
MONO (10^9/L)
At necropsy of the dosing period	5	0.09 ± 0.03	5	0.12 ± 0.10	5	0.08 ± 0.03	5	0.08 ± 0.02
At necropsy of the recovery period	5	0.10 ± 0.02	/	/	/	/	5	0.10 ± 0.05
EOS (10^9/L)
At necropsy of the dosing period	5	0.046 ± 0.011	5	0.064 ± 0.022	5	0.062 ± 0.016	5	0.062 ± 0.026
At necropsy of the recovery period	5	0.052 ± 0.016	/	/	/	/	5	0.060 ± 0.016
BASO (10^9/L)
At necropsy of the dosing period	5	0.012 ± 0.004	5	0.024 ± 0.009	5	0.016 ± 0.009	5	0.020 ± 0.017
At necropsy of the recovery period	5	0.010 ± 0.000	/	/	/	/	5	0.010 ± 0.007
RBC (10^12/L)
At necropsy of the dosing period	5	7.43 ± 0.55	5	7.20 ± 0.35	5	7.36 ± 0.25	5	7.13 ± 0.40
At necropsy of the recovery period	5	7.25 ± 0.35	/	/	/	/	5	7.33 ± 0.28
HGB (g/L)
At necropsy of the dosing period	5	144 ± 8	5	145 ± 5	5	146 ± 2	5	143 ± 5
At necropsy of the recovery period	5	147 ± 4	/	/	/	/	5	148 ± 4
HCT (%)
At necropsy of the dosing period	5	44.5 ± 2.4	5	44.4 ± 2.2	5	43.9 ± 1.3	5	43.3 ± 0.6
At necropsy of the recovery period	5	41.7 ± 1.4	/	/	/	/	5	42.2 ± 1.1
MCV (fL)
At necropsy of the dosing period	5	60.1 ± 3.2	5	61.6 ± 2.0	5	59.7 ± 2.2	5	60.8 ± 2.6
At necropsy of the recovery period	5	57.6 ± 1.8	/	/	/	/	5	57.5 ± 1.2
MCH (pg)
At necropsy of the dosing period	5	19.5 ± 1.2	5	20.2 ± 0.8	5	19.9 ± 0.8	5	20.1 ± 1.6
At necropsy of the recovery period	5	20.3 ± 0.8	/	/	/	/	5	20.3 ± 0.7
MCHC (g/L)
At necropsy of the dosing period	5	324 ± 3	5	327 ± 5	5	333 ± 11	5	331 ± 14
At necropsy of the recovery period	5	352 ± 4	/	/	/	/	5	353 ± 6
PLT (10^9/L)
At necropsy of the dosing period	5	1080 ± 72	5	1087 ± 120	5	1082 ± 64	5	1084 ± 110
At necropsy of the recovery period	5	1018 ± 103	/	/	/	/	5	912 ± 73
RET % (%)
At necropsy of the dosing period	5	2.06 ± 0.40	5	2.04 ± 0.45	5	1.90 ± 0.26	5	2.30 ± 0.44
At necropsy of the recovery period	5	1.91 ± 0.45	/	/	/	/	5	1.83 ± 0.25
RET (10^12/L)
At necropsy of the dosing period	5	0.152 ± 0.027	5	0.146 ± 0.029	5	0.139 ± 0.017	5	0.166 ± 0.024
At necropsy of the recovery period	5	0.137 ± 0.024	/	/	/	/	5	0.135 ± 0.018
APTT (sec)
At necropsy of the dosing period	5	13.9 ± 1.1	5	13.1 ± 2.0	5	13.9 ± 1.9	5	13.9 ± 1.9
At necropsy of the recovery period	5	15.1 ± 2.1	/	/	/	/	5	13.9 ± 1.3
PT (sec)
At necropsy of the dosing period	5	8.4 ± 0.4	5	8.6 ± 0.4	5	8.7 ± 0.9	5	8.3 ± 0.2
At necropsy of the recovery period	5	8.9 ± 0.2	/	/	/	/	5	9.0 ± 0.4

Notes: * Statistical significance was noted when compared with the control (p≤0.05).

 

Table 4 Effects on Hematology of Females

Parameters Detection time	Control		100 mg/kg bw/daygroup		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
WBC (10^9/L)
At necropsy of the dosing period	5	3.84 ± 0.64	5	3.62 ± 1.06	5	3.26 ± 1.61	5	3.57 ± 0.70
At necropsy of the recovery period	5	3.81 ± 0.57	/	/	/	/	5	4.29 ± 1.50
NEU% (%)
At necropsy of the dosing period	5	13.0 ± 1.8	5	17.7 ± 4.7	5	15.9 ± 4.9	5	16.9 ± 5.4
At necropsy of the recovery period	5	14.8 ± 5.4	/	/	/	/	5	14.5 ± 6.2
LYM% (%)
At necropsy of the dosing period	5	83.6 ± 2.3	5	78.2 ± 4.6	5	80.6 ± 5.4	5	79.5 ± 6.0
At necropsy of the recovery period	5	80.4 ± 6.7	/	/	/	/	5	82.0 ± 6.0
MONO% (%)
At necropsy of the dosing period	5	1.3 ± 0.3	5	1.8 ± 0.5	5	1.4 ± 0.3	5	1.4 ± 0.6
At necropsy of the recovery period	5	1.6 ± 0.8	/	/	/	/	5	1.4 ± 0.5
EOS% (%)
At necropsy of the dosing period	5	1.12 ± 0.52	5	1.30 ± 0.10	5	1.24 ± 0.19	5	1.24 ± 0.67
At necropsy of the recovery period	5	2.02 ± 1.11	/	/	/	/	5	1.18 ± 0.45
BASO% (%)
At necropsy of the dosing period	5	0.24 ± 0.11	5	0.24 ± 0.11	5	0.18 ± 0.11	5	0.16 ± 0.05
At necropsy of the recovery period	5	0.20 ± 0.07	/	/	/	/	5	0.14 ± 0.11
NEU (10^9/L)
At necropsy of the dosing period	5	0.49 ± 0.06	5	0.61 ± 0.05	5	0.57 ± 0.47	5	0.59 ± 0.19
At necropsy of the recovery period	5	0.55 ± 0.15	/	/	/	/	5	0.60 ± 0.26
LYM (10^9/L)
At necropsy of the dosing period	5	3.22 ± 0.60	5	2.87 ± 1.00	5	2.57 ± 1.08	5	2.87 ± 0.72
At necropsy of the recovery period	5	3.09 ± 0.69	/	/	/	/	5	3.54 ± 1.35
MONO (10^9/L)
At necropsy of the dosing period	5	0.05 ± 0.01	5	0.07 ± 0.03	5	0.05 ± 0.03	5	0.05 ± 0.01
At necropsy of the recovery period	5	0.06 ± 0.02	/	/	/	/	5	0.05 ± 0.01
EOS (10^9/L)
At necropsy of the dosing period	5	0.042 ± 0.019	5	0.048 ± 0.013	5	0.044 ± 0.028	5	0.040 ± 0.012
At necropsy of the recovery period	5	0.074 ± 0.030	/	/	/	/	5	0.048 ± 0.011
BASO (10^9/L)
At necropsy of the dosing period	5	0.010 ± 0.007	5	0.010 ± 0.007	5	0.004 ± 0.005	5	0.004 ± 0.005
At necropsy of the recovery period	5	0.008 ± 0.004	/	/	/	/	5	0.006 ± 0.009
RBC (10^12/L)
At necropsy of the dosing period	5	7.03 ± 0.45	5	6.84 ± 0.22	5	6.87 ± 0.12	5	6.76 ± 0.16
At necropsy of the recovery period	5	7.13 ± 0.27	/	/	/	/	5	6.99 ± 0.18
HGB (g/L)
At necropsy of the dosing period	5	137 ± 6	5	134 ± 5	5	137 ± 6	5	134 ± 4
At necropsy of the recovery period	5	141 ± 3	/	/	/	/	5	138 ± 4
HCT (%)
At necropsy of the dosing period	5	41.2 ± 1.7	5	40.6 ± 1.4	5	40.8 ± 1.9	5	39.8 ± 1.2
At necropsy of the recovery period	5	40.2 ± 0.8	/	/	/	/	5	39.1 ± 1.0
MCV (fL)
At necropsy of the dosing period	5	58.6 ± 1.2	5	59.4 ± 1.7	5	59.4 ± 2.0	5	58.9 ± 0.9
At necropsy of the recovery period	5	56.4 ± 2.0	/	/	/	/	5	56.0 ± 0.9
MCH (pg)
At necropsy of the dosing period	5	19.6 ± 0.6	5	19.7 ± 0.5	5	19.9 ± 0.6	5	19.9 ± 0.3
At necropsy of the recovery period	5	19.8 ± 0.8	/	/	/	/	5	19.7 ± 0.6
MCHC (g/L)
At necropsy of the dosing period	5	334 ± 9	5	331 ± 4	5	336 ± 11	5	338 ± 6
At necropsy of the recovery period	5	350 ± 3	/	/	/	/	5	351 ± 6
PLT (10^9/L)
At necropsy of the dosing period	5	1150 ± 161	5	1111 ± 43	5	1061 ± 100	5	1126 ± 63
At necropsy of the recovery period	5	991 ± 156	/	/	/	/	5	961 ± 67
RET % (%)
At necropsy of the dosing period	5	2.03 ± 0.62	5	2.03 ± 0.56	5	2.14 ± 0.29	5	2.16 ± 0.64
At necropsy of the recovery period	5	1.77 ± 0.16	/	/	/	/	5	1.66 ± 0.33
RET (10^12/L)
At necropsy of the dosing period	5	0.142 ± 0.039	5	0.139 ± 0.037	5	0.147 ± 0.020	5	0.146 ± 0.041
At necropsy of the recovery period	5	0.127 ± 0.013	/	/	/	/	5	0.116 ± 0.024
APTT (sec)
At necropsy of the dosing period	5	13.2 ± 1.2	5	12.2 ± 1.8	5	12.2 ± 1.7	5	12.1 ± 1.1
At necropsy of the recovery period	5	13.7 ± 1.1	/	/	/	/	5	12.5 ± 1.8
PT (sec)
At necropsy of the dosing period	5	7.7 ± 0.5	5	7.7 ± 0.6	5	7.6 ± 0.3	5	7.7 ± 0.3
At necropsy of the recovery period	5	7.9 ± 0.6	/	/	/	/	5	8.0 ± 0.4

 

Table 5 Effects on Clinical Chemistry of Males

Parameters Detection time	Control		100 mg/kg bw/day group		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
ALB (g/L)
At necropsy of the dosing period	5	38.8 ± 1.2	5	38.2 ± 0.7	5	38.5 ± 1.9	5	38.3 ± 1.0
At necropsy of the recovery period	5	37.4 ± 0.9	/	/	/	/	5	38.3 ± 1.6
TP (g/L)
At necropsy of the dosing period	5	56.8 ± 1.5	5	55.3 ± 1.9	5	55.2 ± 1.6	5	55.6 ± 0.4
At necropsy of the recovery period	5	53.9 ± 0.3	/	/	/	/	5	55.3 ± 1.7
AST (U/L)
At necropsy of the dosing period	5	105.8 ± 15.2	5	107.4 ± 19.8	5	105.6 ± 7.8	5	102.3 ± 21.5
At necropsy of the recovery period	5	93.6 ± 5.7	/	/	/	/	5	88.1 ± 8.4
ALT (U/L)
At necropsy of the dosing period	5	32.2 ± 3.0	5	31.8 ± 6.8	5	34.6 ± 7.7	5	30.4 ± 7.4
At necropsy of the recovery period	5	30.6 ± 1.7	/	/	/	/	5	39.8 ± 9.0
TBIL (μmol/L)
At necropsy of the dosing period	5	0.8 ± 0.1	5	0.7 ± 0.4	5	1.0 ± 0.2	5	0.6 ± 0.1
At necropsy of the recovery period	5	1.0 ± 0.2	/	/	/	/	5	1.7 ± 0.4 *
CK (U/L)
At necropsy of the dosing period	5	676 ± 222	5	704 ± 260	5	652 ± 227	5	582 ± 141
At necropsy of the recovery period	5	439 ± 65	/	/	/	/	5	458 ± 225
CHOL (mmol/L)
At necropsy of the dosing period	5	1.30 ± 0.17	5	1.36 ± 0.37	5	1.08 ± 0.22	5	1.33 ± 0.35
At necropsy of the recovery period	5	1.29 ± 0.27	/	/	/	/	5	1.41 ± 0.38
Ca (mmol/L)
At necropsy of the dosing period	5	2.60 ± 0.03	5	2.59 ± 0.06	5	2.60 ± 0.08	5	2.59 ± 0.07
At necropsy of the recovery period	5	2.52 ± 0.05	/	/	/	/	5	2.57 ± 0.03
TG (mmol/L)
At necropsy of the dosing period	5	0.75 ± 0.22	5	0.72 ± 0.19	5	0.40 ± 0.09 *	5	0.49 ± 0.05
At necropsy of the recovery period	5	0.69 ± 0.19	/	/	/	/	5	0.67 ± 0.21
Crea (μmol/L)
At necropsy of the dosing period	5	18.8 ± 2.2	5	18.0 ± 2.5	5	17.4 ± 1.1	5	19.6 ± 3.3
At necropsy of the recovery period	5	19.6 ± 2.6	/	/	/	/	5	20.2 ± 0.8
GLU (mmol/L)
At necropsy of the dosing period	5	6.85 ± 0.29	5	7.14 ± 0.57	5	6.26 ± 0.38	5	7.08 ± 0.67
At necropsy of the recovery period	5	6.48 ± 0.45	/	/	/	/	5	7.31 ± 1.08
Urea (mmol/L)
At necropsy of the dosing period	5	4.72 ± 0.41	5	4.70 ± 0.66	5	4.67 ± 0.63	5	4.80 ± 1.02
At necropsy of the recovery period	5	4.32 ± 0.51	/	/	/	/	5	4.87 ± 0.64
P (mmol/L)
At necropsy of the dosing period	5	2.76 ± 0.09	5	2.72 ± 0.09	5	2.73 ± 0.20	5	2.95 ± 0.15
At necropsy of the recovery period	5	2.30 ± 0.08	/	/	/	/	5	2.46 ± 0.14
ALP (U/L)
At necropsy of the dosing period	5	150.3 ± 25.1	5	149.2 ± 22.0	5	165.3 ± 45.0	5	164.8 ± 34.3
At necropsy of the recovery period	5	123.3 ± 18.6	/	/	/	/	5	137.9 ± 35.7
LDH (U/L)
At necropsy of the dosing period	5	1137 ± 333	5	1128 ± 388	5	1087 ± 340	5	1060 ± 352
At necropsy of the recovery period	5	868 ± 160	/	/	/	/	5	654 ± 159
K^+(mmol/L)
At necropsy of the dosing period	5	4.56 ± 0.28	5	4.66 ± 0.16	5	4.51 ± 0.04	5	4.53 ± 0.12
At necropsy of the recovery period	5	4.49 ± 0.15	/	/	/	/	5	4.44 ± 0.12
Na^+(mmol/L)
At necropsy of the dosing period	5	143 ± 1	5	143 ± 0	5	144 ± 1	5	144 ± 1
At necropsy of the recovery period	5	144 ± 2	/	/	/	/	5	142 ± 1
Cl^-(mmol/L)
At necropsy of the dosing period	5	101.8 ± 1.2	5	102.8 ± 0.5	5	102.3 ± 1.1	5	101.9 ± 0.7
At necropsy of the recovery period	5	101.4 ± 1.3	/	/	/	/	5	100.6 ± 1.3
A/G
At necropsy of the dosing period	5	2.16 ± 0.10	5	2.26 ± 0.20	5	2.32 ± 0.18	5	2.22 ± 0.17
At necropsy of the recovery period	5	2.29 ± 0.19	/	/	/	/	5	2.25 ± 0.18

Notes: * Statistical significance was noted when compared with the control (p≤0.05).

 

Table 6 Effects on Clinical Chemistry of Females

Parameters Detection time	Control		100 mg/kg bw/day group		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
ALB (g/L)
At necropsy of the dosing period	5	43.9 ± 3.5	5	44.7 ± 1.5	5	42.9 ± 2.7	5	43.6 ± 2.2
At necropsy of the recovery period	5	46.1 ± 3.3	/	/	/	/	5	42.4 ± 4.7
TP (g/L)
At necropsy of the dosing period	5	61.3 ± 4.0	5	59.5 ± 2.4	5	58.3 ± 1.8	5	58.2 ± 3.6
At necropsy of the recovery period	5	61.9 ± 3.8	/	/	/	/	5	57.6 ± 4.0
AST (U/L)
At necropsy of the dosing period	5	105.8 ± 37.2	5	99.8 ± 16.3	5	105.5 ± 3.2	5	97.9 ± 11.8
At necropsy of the recovery period	5	93.3 ± 14.1	/	/	/	/	5	93.5 ± 18.7
ALT (U/L)
At necropsy of the dosing period	5	21.8 ± 4.0	5	25.2 ± 7.2	5	25.0 ± 5.7	5	25.8 ± 7.7
At necropsy of the recovery period	5	28.0 ± 7.0	/	/	/	/	5	29.6 ± 6.1
TBIL (μmol/L)
At necropsy of the dosing period	5	1.1 ± 0.2	5	1.1 ± 0.6	5	1.1 ± 0.5	5	1.5 ± 0.3
At necropsy of the recovery period	5	1.2 ± 0.3	/	/	/	/	5	0.9 ± 0.1
CK (U/L)
At necropsy of the dosing period	5	763 ± 537	5	555 ± 178	5	658 ± 95	5	526 ± 71
At necropsy of the recovery period	5	486 ± 174	/	/	/	/	5	435 ± 142
CHOL (mmol/L)
At necropsy of the dosing period	5	1.50 ± 0.29	5	1.53 ± 0.36	5	1.56 ± 0.59	5	1.55 ± 0.42
At necropsy of the recovery period	5	1.55 ± 0.09	/	/	/	/	5	1.40 ± 0.20
Ca (mmol/L)
At necropsy of the dosing period	5	2.49 ± 0.06	5	2.56 ± 0.06	5	2.50 ± 0.06	5	2.58 ± 0.07
At necropsy of the recovery period	5	2.62 ± 0.06	/	/	/	/	5	2.60 ± 0.03
TG (mmol/L)
At necropsy of the dosing period	5	0.34 ± 0.17	5	0.24 ± 0.07	5	0.23 ± 0.11	5	0.24 ± 0.08
At necropsy of the recovery period	5	0.31 ± 0.06	/	/	/	/	5	0.45 ± 0.30
Crea (μmol/L)
At necropsy of the dosing period	5	26.0 ± 2.3	5	26.4 ± 4.7	5	24.8 ± 2.9	5	25.0 ± 2.5
At necropsy of the recovery period	5	28.0 ± 4.2	/	/	/	/	5	31.8 ± 5.3
GLU (mmol/L)
At necropsy of the dosing period	5	5.90 ± 0.15	5	6.40 ± 0.64	5	6.85 ± 0.27 *	5	6.41 ± 0.20 *
At necropsy of the recovery period	5	6.55 ± 0.14	/	/	/	/	5	6.77 ± 0.54
Urea (mmol/L)
At necropsy of the dosing period	5	4.82 ± 0.48	5	4.64 ± 0.41	5	5.11 ± 0.97	5	5.06 ± 0.37
At necropsy of the recovery period	5	5.32 ± 0.64	/	/	/	/	5	6.38 ± 1.61
P (mmol/L)
At necropsy of the dosing period	5	2.11 ± 0.11	5	2.19 ± 0.22	5	2.17 ± 0.14	5	2.54 ± 0.13 *
At necropsy of the recovery period	5	1.88 ± 0.22	/	/	/	/	5	2.05 ± 0.34
ALP (U/L)
At necropsy of the dosing period	5	72.5 ± 17.6	5	73.7 ± 17.2	5	72.2 ± 16.5	5	84.1 ± 33.1
At necropsy of the recovery period	5	44.9 ± 11.7	/	/	/	/	5	57.4 ± 12.0
LDH (U/L)
At necropsy of the dosing period	5	1093 ± 629	5	1019 ± 328	5	1150 ± 288	5	984 ± 180
At necropsy of the recovery period	5	934 ± 323	/	/	/	/	5	878 ± 311
K^+(mmol/L)
At necropsy of the dosing period	5	4.13 ± 0.19	5	4.14 ± 0.25	5	3.95 ± 0.13	5	4.10 ± 0.22
At necropsy of the recovery period	5	3.98 ± 0.25	/	/	/	/	5	4.00 ± 0.29
Na^+(mmol/L)
At necropsy of the dosing period	5	143 ± 1	5	143 ± 1	5	143 ± 1	5	144 ± 1
At necropsy of the recovery period	5	142 ± 1	/	/	/	/	5	143 ± 1
Cl^-(mmol/L)
At necropsy of the dosing period	5	102.7 ± 1.6	5	103.3 ± 1.2	5	103.7 ± 1.2	5	103.8 ± 0.7
At necropsy of the recovery period	5	101.1 ± 0.5	/	/	/	/	5	101.8 ± 1.2
A/G
At necropsy of the dosing period	5	2.54 ± 0.28	5	3.03 ± 0.17	5	2.81 ± 0.39	5	3.04 ± 0.43
At necropsy of the recovery period	5	2.91 ± 0.27	/	/	/	/	5	2.80 ± 0.45

 

Table 7 Effects of D-Prism via Oral Gavage on Urine parameters of Males

Parameters	Detection time Grade	Control	100 mg/kg bw/day group	300 mg/kg bw/day group	1000 mg/kg bw/day group
Urine color
	Day 28
	Yellow	5	5	5	5
	Recovery day 14
	Yellow	5	/	/	4
	Dull yellow	0	/	/	1
Urine clarity
	Day 28
	Clear	4	2	4	2
	Turbidness	1	3	1	3
	Recovery day 14
	Clear	2	/	/	1
	Turbidness	3	/	/	4
GLU(mmol/L)
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
BIL
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
KET(mmol/L)
	Day 28
	-	5	4	5	4
	1.5	0	1	0	1
	Recovery day 14
	-	2	/	/	4
	1.5	3	/	/	1
SG
	Day 28
	<=1.005	0	0	2	0
	1.010	1	2	0	0
	1.015	1	2	1	5
	1.020	2	1	2	0
	1.025	1	0	0	0
	Recovery day 14
	<=1.005	1	/	/	0
	1.010	0	/	/	1
	1.015	2	/	/	1
	1.020	0	/	/	1
	1.025	1	/	/	0
	>1.030	1	/	/	2
BLO(Ery/μL)
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
pH
	Day 28
	7.5	0	1	1	0
	8.0	4	4	3	4
	8.5	1	0	1	1
	Recovery day 14
	6.0	1	/	/	1
	8.0	2	/	/	0
	8.5	2	/	/	4
PRO(g/L)
	Day 28
	-	5	5	4	3
	0.3	0	0	1	2
	Recovery day 14
	-	2	/	/	3
	0.3	3	/	/	2
URO (μmol/L)
	Day 28
	3.2	5	5	5	5
	Recovery day 14
	3.2	5	/	/	5
NIT
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	4
	+	0	/	/	1
WBC(Leu/μL)
	Day 28
	-	4	5	5	5
	15	1	0	0	0
	Recovery day 14
	-	5	/	/	3
	15	0	/	/	2

Notes: * Statistical significance was noted when compared with the control (P≤0.05).

 

Table 8 Effects of D-Prism via Oral Gavage on Urine parameters of Females

Parameters	Detection time Grade	Control	100 mg/kg bw/day group	300 mg/kg bw/day group	1000 mg/kg bw/day group
Urine color
	Day 28
	Yellow	5	5	5	5
	Recovery day 14
	Yellow	5	/	/	5
Urine clarity
	Day 28
	Clear	5	5	5	5
	Recovery day 14
	Clear	4	/	/	4
	Turbidness	0	/	/	1
	Dirty	1	/	/	0
GLU(mmol/L)
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
BIL
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
KET(mmol/L)
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
SG
	Day 28
	<=1.005	2	2	2	1
	1.010	2	3	3	0
	1.015	1	0	0	3
	1.020	0	0	0	1
	Recovery day 14
	<=1.005	1	/	/	2
	1.010	2	/	/	1
	1.015	1	/	/	1
	1.020	0	/	/	1
	1.025	1	/	/	0
BLO(Ery/μL)
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
pH
	Day 28
	7.5	0	0	1	1
	8.0	4	4	3	4
	8.5	1	1	1	0
	Recovery day 14
	6.5	1	/	/	1
	7.0	0	/	/	2
	7.5	1	/	/	0
	8.0	3	/	/	1
	8.5	0	/	/	1
PRO(g/L)
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	5
URO (μmol/L)
	Day 28
	3.2	5	5	5	5
	Recovery day 14
	3.2	5	/	/	5
NIT
	Day 28
	-	4	4	4	4
	+	1	1	1	1
	Recovery day 14
	-	4	/	/	4
	+	1	/	/	1
WBC(Leu/μL)
	Day 28
	-	5	5	5	5
	Recovery day 14
	-	5	/	/	4
	15	0	/	/	1

 

Table 9 Effects on Organ Weight and Ratios of Males

Parameters Detection time	Control		100 mg/kg bw/day group		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
Terminal Body weight (g)
End of Dosingperiod	5	385.860 ± 21.770	5	387.180 ± 31.442	5	364.880 ± 17.616	5	375.640 ± 25.621
End of recovery period	5	410.240 ± 50.549	/	/	/	/	5	436.600 ± 51.849
Organ weight (g)
Thyroid and parathyroid glands
End of Dosing period	5	0.0238 ± 0.0036	5	0.0254 ± 0.0036	5	0.0242 ± 0.0045	5	0.0193 ± 0.0049
End of recovery period	5	0.0292 ± 0.0074	/	/	/	/	5	0.0326 ± 0.0043
Liver
End of Dosing period	5	11.433 ± 1.055	5	11.611 ± 1.741	5	10.327 ± 1.220	5	10.679 ± 0.960
End of recovery period	5	11.171 ± 1.270	/	/	/	/	5	12.166 ± 1.971
Spleen
End of Dosing period	5	0.772 ± 0.084	5	0.857 ± 0.184	5	0.852 ± 0.077	5	0.804 ± 0.110
End of recovery period	5	0.715 ± 0.099	/	/	/	/	5	0.855 ± 0.115
Kidney
End of Dosing period	5	2.670 ± 0.153	5	2.765 ± 0.350	5	2.705 ± 0.165	5	2.534 ± 0.295
End of recovery period	5	2.904 ± 0.363	/	/	/	/	5	2.963 ± 0.324
Adrenal gland
End of Dosing period	5	0.057 ± 0.005	5	0.056 ± 0.004	5	0.061 ± 0.011	5	0.048 ± 0.009
End of recovery period	5	0.049 ± 0.009	/	/	/	/	5	0.053 ± 0.008
Testis
End of Dosing period	5	2.846 ± 0.843	5	2.809 ± 1.175	5	3.251 ± 0.207	5	3.112 ± 0.299
End of recovery period	5	3.368 ± 0.156	/	/	/	/	5	3.200 ± 0.278
Epididymis
End of Dosing period	5	0.981 ± 0.089	5	0.897 ± 0.196	5	0.946 ± 0.109	5	0.910 ± 0.074
End of recovery period	5	1.176 ± 0.057	/	/	/	/	5	1.183 ± 0.097
Thymus gland
End of Dosing period	5	0.605 ± 0.112	5	0.632 ± 0.117	5	0.537 ± 0.074	5	0.591 ± 0.202
End of recovery period	5	0.489 ± 0.135	/	/	/	/	5	0.456 ± 0.134
Heart
End of Dosing period	5	1.277 ± 0.110	5	1.427 ± 0.391	5	1.333 ± 0.114	5	1.247 ± 0.101
End of recovery period	5	1.377 ± 0.161	/	/	/	/	5	1.446 ± 0.184
Brain
End of Dosing period	5	2.031 ± 0.084	5	1.972 ± 0.097	5	1.975 ± 0.013	5	1.937 ± 0.102
End of recovery period	5	1.980 ± 0.122	/	/	/	/	5	2.054 ± 0.080
Prostate + seminal vesicle + coagulation gland
End of Dosing period	5	2.295 ± 0.253	5	2.102 ± 0.215	5	2.290 ± 0.351	5	2.117 ± 0.191
End of recovery period	5	2.996 ± 0.302					5	2.744 ± 0.383
Organ/Body Weight Ratio (%)
Thyroid and parathyroid glands
End of Dosing period	5	0.0061 ± 0.0006	5	0.0066 ± 0.0013	5	0.0066 ± 0.0013	5	0.0051 ± 0.0013
End of recovery period	5	0.0071 ± 0.0015	/	/	/	/	5	0.0076 ± 0.0018
Liver
End of Dosing period	5	2.962 ± 0.200	5	2.989 ± 0.284	5	2.826 ± 0.229	5	2.843 ± 0.177
End of recovery period	5	2.727 ± 0.089	/	/	/	/	5	2.781 ± 0.258
Spleen
End of Dosing period	5	0.200 ± 0.025	5	0.220 ± 0.037	5	0.234 ± 0.021	5	0.215 ± 0.032
End of recovery period	5	0.177 ± 0.032	/	/	/	/	5	0.199 ± 0.044
Kidney
End of Dosing period	5	0.692 ± 0.029	5	0.713 ± 0.042	5	0.741 ± 0.025	5	0.673 ± 0.046
End of recovery period	5	0.710 ± 0.069	/	/	/	/	5	0.680 ± 0.018
Adrenal gland
End of Dosing period	5	0.015 ± 0.002	5	0.015 ± 0.002	5	0.017 ± 0.003	5	0.013 ± 0.003
End of recovery period	5	0.012 ± 0.002	/	/	/	/	5	0.012 ± 0.002
Testis
End of Dosing period	5	0.735 ± 0.204	5	0.721 ± 0.289	5	0.891 ± 0.020	5	0.831 ± 0.085
End of recovery period	5	0.832 ± 0.118	/	/	/	/	5	0.739 ± 0.082
Epididymis
End of Dosing period	5	0.254 ± 0.016	5	0.233 ± 0.055	5	0.259 ± 0.022	5	0.243 ± 0.017
End of recovery period	5	0.290 ± 0.034	/	/	/	/	5	0.275 ± 0.044
Thymus gland
End of Dosing period	5	0.156 ± 0.023	5	0.163 ± 0.029	5	0.147 ± 0.018	5	0.156 ± 0.045
End of recovery period	5	0.118 ± 0.021	/	/	/	/	5	0.104 ± 0.023
Heart
End of Dosing period	5	0.331 ± 0.014	5	0.366 ± 0.083	5	0.365 ± 0.024	5	0.332 ± 0.023
End of recovery period	5	0.336 ± 0.020	/	/	/	/	5	0.331 ± 0.006
Brain
End of Dosing period	5	0.528 ± 0.039	5	0.511 ± 0.027	5	0.542 ± 0.026	5	0.516 ± 0.019
End of recovery period	5	0.488 ± 0.056	/	/	/	/	5	0.476 ± 0.056
Prostate + seminal vesicle + coagulation gland
End of Dosing period	5	0.596 ± 0.071	5	0.543 ± 0.026	5	0.626 ± 0.071	5	0.565 ± 0.050
End of recovery period	5	0.735 ± 0.072	/	/	/	/	5	0.633 ± 0.095
Organ/Brain Weight Ratio (%)
Thyroid and parathyroid glands
End of Dosing period	5	1.174 ± 0.206	5	1.293 ± 0.196	5	1.227 ± 0.223	5	0.999 ± 0.265
End of recovery period	5	1.469 ± 0.363	/	/	/	/	5	1.583 ± 0.191
Liver
End of Dosing period	5	563.56 ± 55.53	5	588.41 ± 81.37	5	523.15 ± 63.32	5	551.35 ± 43.01
End of recovery period	5	564.14 ± 56.55	/	/	/	/	5	591.17 ± 84.69
Spleen
End of Dosing period	5	37.98 ± 3.77	5	43.30 ± 8.26	5	43.14 ± 3.83	5	41.55 ± 5.81
End of recovery period	5	36.08 ± 4.06	/	/	/	/	5	41.59 ± 5.30
Kidney
End of Dosing period	5	131.68 ± 10.62	5	139.85 ± 11.34	5	137.03 ± 8.71	5	130.57 ± 10.20
End of recovery period	5	146.27 ± 11.58	/	/	/	/	5	144.26 ± 15.15
Adrenal gland
End of Dosing period	5	2.82 ± 0.36	5	2.84 ± 0.30	5	3.08 ± 0.54	5	2.48 ± 0.54
End of recovery period	5	2.47 ± 0.41	/	/	/	/	5	2.59 ± 0.34
Testis
End of Dosing period	5	141.05 ± 43.73	5	141.01 ± 56.59	5	164.66 ± 10.67	5	160.62 ± 12.05
End of recovery period	5	170.47 ± 10.68	/	/	/	/	5	156.18 ± 17.65
Epididymis
End of Dosing period	5	48.43 ± 5.67	5	45.37 ± 9.38	5	47.92 ± 5.64	5	46.93 ± 2.19
End of recovery period	5	59.42 ± 1.67	/	/	/	/	5	57.68 ± 5.86
Thymus gland
End of Dosing period	5	29.90 ± 6.22	5	32.10 ± 6.42	5	27.22 ± 3.84	5	30.44 ± 9.85
End of recovery period	5	24.85 ± 7.58	/	/	/	/	5	22.02 ± 5.73
Heart
End of Dosing period	5	63.00 ± 6.98	5	71.93 ± 16.98	5	67.52 ± 6.07	5	64.32 ± 2.83
End of recovery period	5	69.56 ± 7.49	/	/	/	/	5	70.30 ± 7.77
Prostate + seminal vesicle + coagulation gland
End of Dosing period	5	113.31 ± 15.53	5	106.41 ± 6.45	5	115.97 ± 17.93	5	109.34 ± 8.80
End of recovery period	5	151.15 ± 9.34	/	/	/	/	5	133.66 ± 19.02

Notes: * Statistical significance was noted when compared with the control (p≤0.05).

 

Table 10 Effects of D-Prism via Oral Gavage on Organ Weight and Ratios of Females

Parameters Detection time	Control		100 mg/kg bw/day group		300 mg/kg bw/day group		1000 mg/kg bw/day group
	n	Mean± SD	n	Mean± SD	n	Mean± SD	n	Mean± SD
Terminal Body weight (g)
End of Dosing period	5	228.660 ± 15.507	5	230.340 ± 16.748	5	223.860 ± 20.493	5	227.640 ± 18.307
End of recovery period	5	228.840 ± 26.364	/	/	/	/	5	228.900 ± 11.755
Organ weight (g)
Thyroid and parathyroid glands
End of Dosing period	5	0.0189 ± 0.0046	5	0.0192 ± 0.0029	5	0.0245 ± 0.0039	5	0.0195 ± 0.0063
End of recovery period	5	0.0299 ± 0.0120	/	/	/	/	5	0.0221 ± 0.0057
Liver
End of Dosing period	5	6.431 ± 0.699	5	6.350 ± 0.492	5	6.614 ± 0.648	5	6.044 ± 0.307
End of recovery period	5	6.192 ± 0.787	/	/	/	/	5	6.128 ± 0.568
Spleen
End of Dosing period	5	0.646 ± 0.212	5	0.572 ± 0.135	5	0.673 ± 0.194	5	0.622 ± 0.065
End of recovery period	5	0.540 ± 0.119	/	/	/	/	5	0.547 ± 0.071
Kidney
End of Dosing period	5	1.739 ± 0.095	5	1.698 ± 0.148	5	1.723 ± 0.121	5	1.666 ± 0.185
End of recovery period	5	1.633 ± 0.309	/	/	/	/	5	1.619 ± 0.052
Adrenal gland
End of Dosing period	5	0.062 ± 0.004	5	0.064 ± 0.008	5	0.062 ± 0.009	5	0.059 ± 0.009
End of recovery period	5	0.061 ± 0.005	/	/	/	/	5	0.055 ± 0.009
Ovary
End of Dosing period	5	0.102 ± 0.009	5	0.099 ± 0.012	5	0.104 ± 0.020	5	0.097 ± 0.016
End of recovery period	5	0.092 ± 0.011	/	/	/	/	5	0.085 ± 0.011
Uterus
End of Dosing period	5	0.616 ± 0.147	5	0.567 ± 0.127	5	0.545 ± 0.097	5	0.578 ± 0.239
End of recovery period	5	0.447 ± 0.071	/	/	/	/	5	0.548 ± 0.167
Thymus gland
End of Dosing period	5	0.470 ± 0.124	5	0.412 ± 0.060	5	0.445 ± 0.067	5	0.400 ± 0.119
End of recovery period	5	0.417 ± 0.029	/	/	/	/	5	0.349 ± 0.053 *
Heart
End of Dosing period	5	0.854 ± 0.068	5	0.876 ± 0.075	5	0.859 ± 0.155	5	0.840 ± 0.132
End of recovery period	5	0.865 ± 0.111	/	/	/	/	5	0.822 ± 0.077
Brain
End of Dosing period	5	1.823 ± 0.097	5	1.822 ± 0.106	5	1.855 ± 0.071	5	1.896 ± 0.055
End of recovery period	5	1.813 ± 0.062	/	/	/	/	5	1.816 ± 0.094
Organ/Body Weight Ratio (%)
Thyroid and parathyroid glands
End of Dosing period	5	0.0082 ± 0.0015	5	0.0084 ± 0.0012	5	0.0110 ± 0.0016	5	0.0086 ± 0.0027
End of recovery period	5	0.0129 ± 0.0049	/	/	/	/	5	0.0097 ± 0.0028
Liver
End of Dosing period	5	2.807 ± 0.122	5	2.758 ± 0.113	5	2.960 ± 0.230	5	2.662 ± 0.125
End of recovery period	5	2.709 ± 0.213	/	/	/	/	5	2.679 ± 0.228
Spleen
End of Dosing period	5	0.286 ± 0.102	5	0.247 ± 0.044	5	0.297 ± 0.057	5	0.275 ± 0.036
End of recovery period	5	0.236 ± 0.043	/	/	/	/	5	0.238 ± 0.021
Kidney
End of Dosing period	5	0.761 ± 0.028	5	0.737 ± 0.028	5	0.771 ± 0.032	5	0.731 ± 0.039
End of recovery period	5	0.710 ± 0.067	/	/	/	/	5	0.708 ± 0.021
Adrenal gland
End of Dosing period	5	0.027 ± 0.002	5	0.028 ± 0.005	5	0.028 ± 0.005	5	0.026 ± 0.003
End of recovery period	5	0.027 ± 0.004	/	/	/	/	5	0.024 ± 0.004
Ovary
End of Dosing period	5	0.045 ± 0.005	5	0.043 ± 0.006	5	0.047 ± 0.009	5	0.043 ± 0.008
End of recovery period	5	0.041 ± 0.004	/	/	/	/	5	0.037 ± 0.005
Uterus
End of Dosing period	5	0.271 ± 0.073	5	0.245 ± 0.050	5	0.247 ± 0.056	5	0.253 ± 0.097
End of recovery period	5	0.195 ± 0.022	/	/	/	/	5	0.238 ± 0.065
Thymus gland
End of Dosing period	5	0.208 ± 0.061	5	0.179 ± 0.025	5	0.199 ± 0.028	5	0.174 ± 0.042
End of recovery period	5	0.184 ± 0.015	/	/	/	/	5	0.153 ± 0.022 *
Heart
End of Dosing period	5	0.374 ± 0.033	5	0.381 ± 0.032	5	0.382 ± 0.043	5	0.368 ± 0.034
End of recovery period	5	0.379 ± 0.039	/	/	/	/	5	0.359 ± 0.021
Brain
End of Dosing period	5	0.799 ± 0.054	5	0.793 ± 0.060	5	0.834 ± 0.071	5	0.837 ± 0.072
End of recovery period	5	0.800 ± 0.083	/	/	/	/	5	0.795 ± 0.047
Organ/Brain Weight Ratio (%)
Thyroid and parathyroid glands
End of Dosing period	5	1.034 ± 0.211	5	1.053 ± 0.128	5	1.326 ± 0.235	5	1.036 ± 0.359
End of recovery period	5	1.638 ± 0.649	/	/	/	/	5	1.217 ± 0.304
Liver
End of Dosing period	5	352.67 ± 31.78	5	349.18 ± 28.85	5	356.34 ± 29.81	5	319.16 ± 22.59
End of recovery period	5	341.10 ± 39.02	/	/	/	/	5	337.94 ± 33.09
Spleen
End of Dosing period	5	35.25 ± 10.38	5	31.44 ± 7.26	5	36.20 ± 10.06	5	32.88 ± 4.21
End of recovery period	5	29.65 ± 5.83	/	/	/	/	5	30.16 ± 4.09
Kidney
End of Dosing period	5	95.48 ± 5.64	5	93.46 ± 9.96	5	92.93 ± 6.23	5	87.98 ± 10.84
End of recovery period	5	89.92 ± 16.23	/	/	/	/	5	89.29 ± 5.25
Adrenal gland
End of Dosing period	5	3.38 ± 0.27	5	3.52 ± 0.61	5	3.33 ± 0.56	5	3.13 ± 0.48
End of recovery period	5	3.34 ± 0.31	/	/	/	/	5	3.00 ± 0.36
Ovary
End of Dosing period	5	5.60 ± 0.59	5	5.45 ± 0.88	5	5.62 ± 1.01	5	5.15 ± 0.90
End of recovery period	5	5.10 ± 0.60	/	/	/	/	5	4.68 ± 0.79
Uterus
End of Dosing period	5	33.95 ± 8.87	5	31.15 ± 6.79	5	29.34 ± 4.92	5	30.38 ± 12.26
End of recovery period	5	24.65 ± 3.89	/	/	/	/	5	30.25 ± 9.62
Thymus gland
End of Dosing period	5	26.07 ± 8.01	5	22.62 ± 3.05	5	23.91 ± 2.97	5	21.12 ± 6.33
End of recovery period	5	23.03 ± 1.68	/	/	/	/	5	19.28 ± 3.06 *
Heart
End of Dosing period	5	46.90 ± 4.39	5	48.18 ± 4.35	5	46.25 ± 7.68	5	44.29 ± 6.54
End of recovery period	5	47.65 ± 5.65	/	/	/	/	5	45.31 ± 4.38

 

Conclusions:

As no adverse effects were observed up to the highest dose group the NOAEL was considered to be >= 1000 mg/kg bw/day for female and male rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch Code: 1) conducted with the test substance. The study is sufficient to fulfill the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

One repeated dose oral toxicity study was performed for zinc aluminium oxide according to OECD Guidelines for Testing of Chemicals 407, “Repeated Dose 28-Day Oral Toxicity Study in Rodents (2008)” in compliance with GLP (Xu, 2017).

 

In the study, five males and females of SD rats were administered zinc aluminium oxide at the dose level of 0 (control, C), 100 (low dose group, LD), 300 (medium group, MD) and 1000 (high dose group, HD) mg/kg bw/day in water (sterilized injection water) once daily for 28 days via gavage. In addition, a satellite control and a high-dose group of 5 rats/dose /sex was treated in parallel, with a 14-day post-exposure recovery period to assess the reversibility of treatment-related effects. The first day of administration was defined as the Day 1 of treatment and the Day 29 as the Day 1 of recovery.

 

The cage side observations of each group were observed daily both during the treatment and recovery periods and detailed clinical signs were observed and scored prior to administration, weekly during the treatment and the last week of recovery. Grip strength was measured in the last week of dosing and recovery, respectively. Body weight gain and food consumption were determined weekly during treatment and recovery. Urine examinations were conducted in the last week of treatment and recovery, respectively. Blood sampling of all animals for haematological and clinical chemistry examinations and vaginal smear cycle examination in all females were made on the day of termination treatment and recovery prior to sacrificing. 10 animals (half males and half females) of each treatment groups, as well as 10 animals (half males and half females) of each satellite groups were subject to macroscopy, organ weight determination and histopathological examinations.

 

No mortality was observed during the study period. During the treatments and recovery the animals were generally in good health condition, no toxicologically relevant clinical signs were observed in the cage side observations, hand-held observations and open field observations, or the stimulation test. Increased grip strength was observed in females at 1000 mg/kg bw/day in the 4th week of treatment and in males at the end of recovery period (p<0.05). However, there was no statistical significant difference from controls observed on abnormal gripping in males at 100, 300 and 1000 mg/kg bw/day group during treatment in the 4th week, neither in females at 1000 mg/kg bw/day during the 2nd week of the recovery period. Since the difference from control animals was slight and observed on grip strength only, the effect was judged to be incidental rather than treatment related.

 

The body weight, body weight gain and food consumption was comparable between the control group and treatment and recovery groups, respectively.

 

At the end of treatment, haematological examinations did not reveal abnormal changes in any animals at any dose level. The haematological parameters of females and males in the satellite group of 1000 mg/kg bw/day was normal at end of the recovery period. In the clinical chemistry examinations, statistically significant increase levels of glucose (300 mg/kg bw/day and 1000 mg/kg bw/day group), of phosphor (1000 mg/kg bw/day group) were noted in females at the end of the treatment period. In males statisitically significant decreased triglyceride levels were observed at 300 mg/kg bw/day at the end of the treatment period and increased total bilirubin levels at 1000 mg/kg bw/day at the end of recovery period. However, the decrease of triglyceride levels was not dose-related and the difference was slight and thus not considered toxicological relevant. The increase of total bilirubin levels occurred in the recovery period and was within the fluctuation range of animals at the same age in the laboratory (0.37 – 1.8 µmol/L). The increase of glucose and phosphor levels compared to the control group was slight and with the fluctuation range of females at the same age in the laboratory (glucose: 4.59 – 8.32 mmol/L, P: 1.70 – 2.96 mmol//L). Therefore, the observations above are considered to be not toxicological relevant.

 

Statistically significant decreases in thymus weight, thymus/body weight ratio and thymus/brain weight ratio at the end of recovery period (p<0.05) compared to the control group was observed in females at 1000 mg/kg bw/day. However, the differences were slight (<= 16.85%) and appeared in recovery period only. Furthermore, because no statistically significant differences were observed in the dosing period and no organ abnormalities were observed in the histopathological examinations, the observation was considered to be normal test fluctuations and have no toxicological relevance. Slight increase of uterine weight was observed in individual females. This observation was compared with the vaginal smear cycle examination on the day of necropsy. The results indicated that the animals with increased uterus weight were in the emotional period. Thus, this is the normal phenomenon of the estrus cycle, and thus considered not treatment related.

 

No abnormal changes were noted in the results of urinalysis. No statistically significant differences compared to the control group was observed in absolute and relative organ weights in animals at the end of dosing and recovery period (p>0.05), respectively. No abnormal changes were noted in gross pathological and histopathological examinations.

 

Based on the data generated in the study, the NOAEL (No Observed Adverse Effect Level) of zinc aluminium oxide is considered to be greater than 1000 mg/kg body weight/day for the 28-Day repeated dose oral toxicity study in male and female rats.

Justification for classification or non-classification

The available data on repeated dose toxicity indicate that the Zinc Aluminium Oxide does not meet the critiria for classification according to Regulation (EC) No. 1272/2008.