Aluminum magnesium vanadium oxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Nov 2014 - 25 Jan 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

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)

Remarks:

National (CFDA) GLP certificate

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:Experimental Animal Center of Academy of Military Medical Sciences
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 5-7 weeks
- Weight at study initiation: Male 188.8 ± 8.4 g, female 165.7 ± 7.4 g
- Fasting period before study: Non- fasted animals at start of dosing
- Housing: The rats were housed in groups of 5 animals of one sex per cage.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Animals were quarantined for an acclimatization period of seven days.

DETAILS OF FOOD AND WATER QUALITY:
The SPF mouse/rat maintenance diet was provided by the Experimental Animal Center of Academy of Military Medical Sciences (license No: SCXK (Army) 2012-0003). The analyses report of nutrients and contaminants were provided by diet manufacturers in every quarter of year.
The results of representative reports showed that the nutrients and pollutants level was in compliance with national standards (GB 14924.2-2001 Laboratory animals-Hygienic standard for formula feeds and GB 14924.3-2010 Laboratory animals-Nutrients for formula feeds) without interference or impact on the study.
Animal drinking water was filter-sterilized prepared by water filtered machine. Rats had free access to water. The microbes and other indicators of drinking water were analyzed twice a year. The results of representative water quality analytical reports showed that the microbes and pollutant contaminants level was in compliance with national standard (GB5749-2006 Standards for drinking water quality) without interference or impact on study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40-70%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): day/nightcycle was 12 hours (12 hours light from 06:00-18:00, 12 hours dark from 18:00-06:00)

IN-LIFE DATES: From: 21 Nov 2014 To:25 Mar 2015

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item formulations were freshly prepared every day immediately prior to application. Appropriate amount of the test item was weighted into a tared glass vial on a suitable precision balance and the vehicle (corn oil) was added to obtain the designed final concentration of the test item formulations. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before dose administration.
The vehicle (corn oil) was used as the control formulation.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is frequently used as a suitable vehicle in animal experiment when the solubility of test article is low in water. Moreover, coin oil has been proposed as a suitable vehicle by the OECD (OECD Guidelines for Testing of Chemicals: 408 repeated dose 90-Day oral toxicity study in rodents, Sep 1998). Sponsor has agreed to use corn oil as vehicle in this study.
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 0.4 mL/100 g bw
- Lot/batch no. (if required): QS130302011011
- Purity:according to the nactional guidance on maize oil (GB 19111-2003)

Analytical verification of doses or concentrations:

no

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.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily, continuously

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low Dose(LD) group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Medium Dose (MD) group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose(HD) group
high dose with recovery period (HDR) group

No. of animals per sex per dose:

10 aminals per sex per dose in control (C), LD, MD and HD groups
5 animals per sex per dose in satellite group: control recovery (CR) and HDR group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a GLP 28-day repeated dose oral toxicity study in Sprague Dawley rats. The NOAEL of 28-day repeated dose oral toxicity study was 1000 mg/kg bw.

- Post-exposure recovery period in satellite groups: 28 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day during the treatment and recovery periods
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly during the treatment and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Once by animal receiving; once before experimental grouping; weekly during the treatmentand recovery period and once before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly during the treatment and recovery periods
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before administration and at the end of treatment/recovery period.
- Dose groups that were examined: All animals of C, LD, MD, HD, CR and HDR groups before administration; at the end of treatment/recovery period, all animals of C and HD groups are checked firstly, other groups of animals are subsequent checked when C and HD groups have ophthalmologic findings.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period and recovery periods
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters examined: red blood cell (RBC), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), platelet distribution width (PDW), hematocrit (HCT), mean corpuscular volume (MCV), red cell distribution width (RDW), mean platelet volume (MPV), white blood cell (WBC) and classification.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period and recovery period
- Animals fasted: Yes
- How many animals:all
- Parameters examined: aspartate amino transferase (AST), alanine amino transterase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), total protein (TP), albumin (ALB), total bilirubin (TB), direct bilirubin (DB), triglycerides (TG), urea (Urea), total cholesterol (TC), glucose (GLU), creatinine (Cre), sodium (Na+), potassium (K+), chlorine (Cl-)

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of treatment period and recovery period
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: general characters, glucose (Glu), bilirubin (Bil), ketone body (Ket), urinary specific gravity (SG), blood (Bld), pH, urinary protein (Pro), urobilinogen (Uro), nitrite (Nit), white cell (WBC)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly during the treatment and recovery period
- Dose groups that were examined: All groups in the treatment and recovery period
- Battery of functions tested: pupillary reflex, approach response, touch response, air-righting response, pain perception (Tail pinch), startle response

IMMUNOLOGY: No

OTHER: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Sacrifice date: At the terminal of treatment period (26-Mar-2015) and recovery period (23-Apr-2015).
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized and killed by exsanguination.
The following organs from all animals were weighed before fixation and recorded at scheduled necropsy: Brain, Heart, Liver, Spleen, Lung, kidney, Adrenal glands, Thymus, Testis, Epididymis, Uterus and Ovary.
Relative organ weights were calculated on the basis of the body weight. The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios were determined.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered formaldehyde solution.
Heart, Lungs, Liver, Spleen, Kidney, Adrenal glands, Pancreas, Oesophagus, Stomach, Small and large intestines, Lymph nodes, Submandibular gland, Thyroid gland, Parathyroid glands, Trachea, Thymus gland, Testes, Ovaries, Epididymides, Uterus, Prostate, Mammary glands, Brain (cerebrum, cerebellum and pons), Pituitary gland, Sciatic nerve with skeletal muscle, Urinary bladder, Spinal cord, Optic nerve, Thoracic aorta, Sternum with bone marrow.

Statistics:

A statistical analysis of results of body weight, food consumption, parameters of hematology, blood coagulation and clinical biochemistry and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnet t Test. Pathological data were analyzed descriptively and tissue lesions incidence were analyzed using chi-square Test. These statistics were performed with SPSS software (p<0.05 was considered as statistically significant).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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):

Dose-dependent reduction of MPV (fL) (p<0.05) was observed in males, while there were no changes of PLT (109/L) noted when compared to the control group. Dose-dependent reduction of MCV (fL) was noted in females (p<0.05), while there were no changes of RBC (1012/L) observed when compared to the control group. Significantly reduced means of RDW (%) (p<0.05) in females of group LD, and significantly reduced means of PDW (%) and EOS (%) (p<0.05) in females of group HD were noted, respectively. Although some of the differences attained statistical significance (p< 0.05) of the examined parameters, the values of these changes were within historical control data range.
There were no statistically significance changes found in any other measured hematological parameters in treated groups when compared to control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Dose-dependent reduction of TP (g/L) (p<0.01) was observed in males when compared to the control group. Significantly elevated means of Urea (mmol/L) (p<0.05) was noted in males and females of the group HD, but there was no change of CREA (μmol/L) observed. Significantly elevated means of ALT (U/L) (p<0.05) was noted in males of group MD, and significantly reduced means of ALB (g/L) (p<0.05) and GLU (mmol/L) (p<0.01) were noted in males of group HD and HDR, respectively. Significantly reduction of AST (U/L) (p<0.05) was noted in females of group HD. However, the values of these changes remained within the ranges of the historical control data.
There were no statistically significance changes found in any other measured clinical biochemical parameters in treated groups when compared to control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly changes of BIL (p<0.05) was noted in females of group LD. However, there were no significantly changes in TBIL and D-BIL in the clinical biochemistry and therefore the changes of BIL was judged to be of no toxicological significance. There were no statistically or biological significant changes compared to those in the control group in any other examination parameter of treated groups.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational battery was conducted weekly. There were marked changes of the urination and defecation noted in the treatment groups when compared to control group. The changes of the measured urination or defecation did not show a trend of continuous consistency or a dose relationship during the whole study. These inter-group differences of the urination and defecation did not show biological significance and therefore were judged to be variations in measurement during the study.
There was no biologically significant effect observed in any other parameters of functional observational battery in all treated groups during the whole study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute organ weights: the mean thymus gland weight of males in group HD and the mean liver weight of females in group LD was decreased (p< 0.05) when compared to group C of the same sex. The changes only were judged to be have no toxicological significance.
Relative organ weights: there was no significantly change noted in any of the dosed group when compared to group C of the same sex.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All animal survived up to scheduled terminal sacrifice of the administration or recovery period.
Stomach, spleen and abdominal adhesions was observed in one female (R1016) of control group. Nephrohydrosis was found in one male (R4063) of group HD. Visible hard mass of right kidney was observed in one female (R4080) of group HD. All gross lesions noted were single observations. The macroscopic findings were judged to be incidental and not treatment related.
No other changes were detected associated with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Different degree of focal chronic inflammation observed in heart and kidney in control and HD groups. Prostate chronic inflammation was noted in few animals in control, HD and HDR group. These findings were assumed to be unrelated to treatment and judged to be spontaneous occurring background pathology findings in this strain of this age.
At the end of treatment period, local proliferation and focal inflammation lesions focal inflammation in kidney in individual animal was judged to be incidental rather than treatment-related.
There were no test item treated related histopathological changes found in the 90-day repeated dose oral toxicity with Aluminium Magnesium Vanadium Oxide.

Histopathological findings: neoplastic:

no effects observed

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 adverse effects observed

Key result

Critical effects observed:

no

Results:

Table 1. Mean Haematology and Coagulation

Group	Sex	Animal Number	MCV(fL)	RDW (%)	MPV (fL)	PDW (%)
End of Treatment Period
C	M	10	53.1±1.6	13.4±0.5	3.9±0.4	14.8±0.7
	F	10	55.9±1.6	13.1±1.1	4.1±0.5	15.2±0.7
LD	M	10	52.3±2.5	13.1±0.3	3.6±0.5	14.8±0.6
	F	10	54.4±2.0	12.5±0.3*	3.8±0.5	14.9±0.6
MD	M	10	50.7±2.0	13.4±0.3	3.4±0.4*	14.4±0.4
	F	10	54.3±0.9	12.7±0.4	3.8±0.5	14.9±0.6
HD	M	10	51.8±2.3	13.4±0.4	3.2±0.4**	14.3±0.4
	F	10	53.8±1.1*	12.7±0.2	3.1±0.5**	14.3±0.6**
End of Recovery Period
CR	M	5	51.5±1.5	13.5±0.3	3.7±0.4	14.5±0.3
	F	5	55.6±2.8	12.7±0.1	4.1±0.5	15.2±0.6
HDR	M	5	52.1±2.2	13.9±0.2	3.8±0.4	14.9±0.5
	F	5	54.2±1.4	13.0±0.2	3.8±0.3	15.0±0.8

Data showed as Mean ± SD;*p<0.05 and **p<0.01vs C group with the same gender and period.

Table 2. MeanHaematology and Coagulation

Group	Sex	Animal Number	EOS(%)
End of Treatment Period
C	M	10	0.4±0.2
	F	10	0.8±0.7
LD	M	10	0.5±0.2
	F	10	0.7±0.4
MD	M	10	0.7±0.4
	F	10	0.8±0.4
HD	M	10	0.6±0.3
	F	10	0.5±0.2
End of Recovery Period
CR	M	5	0.7±0.5
	F	5	1.3±0.5
HDR	M	5	0.6±0.3
	F	5	0.5±0.2*

Data showed as Mean ± SD;*p<0.05 and **p<0.01vs C group with the same gender and period

Table 3. Mean Clinical Biochemistry

Group	Sex	Animal Number	ALT (U/L)	AST (U/L)	TP (g/L)	ALB (g/L)	GLU (mmol/L)	Urea (mmol/L)
End of Treatment Period
C	M	10	31±4	82±18	58.8±1.2	21.9±0.9	7.97±0.39	4.4±0.8
	F	10	31±5	84±8	64.4±3.3	25.9±2.8	8.28±0.72	4.7±0.6
LD	M	10	36±6	75±16	57.8±2.1	21.9±0.8	7.50±0.74	4.2±0.3
	F	10	35±8	76±8	65.2±3.4	26.2±2.7	8.11±1.16	5.2±1.1
MD	M	10	39±6*	78±9	55.8±2.0**	21.3±0.6	7.82±1.11	5.0±0.6
	F	10	29±7	80±14	64.2±3.8	26.7±2.0	8.19±0.97	5.4±1.1
HD	M	10	38±11	75±9	55.3±2.8**	21.0±0.7*	7.64±0.95	6.3±1.1**
	F	10	28±6	68±22*	60.8±4.5	24.9±2.1	7.85±0.57	5.9±1.1*
End of Recovery Period
CR	M	5	34±3	120±17	61.1±2.4	21.2±0.5	9.26±0.60	5.3±0.3
	F	5	25±3	74±10	68.0±5.2	26.4±2.1	8.56±1.06	5.7±0.6
HDR	M	5	33±4	100±19	59.4±3.1	20.6±1.1	7.73±0.59**	5.2±0.9
	F	5	21±2	65±9	68.8±5.0	26.5±2.2	7.79±0.85	5.7±0.3

Data showed as Mean ± SD;*p<0.05 and **p<0.01vs C group with the same gender and period.

Table 4. MeanUrinalyses

Group	Sex	N	Bil
C	M	5	−: 1/5
			+: 4/5
	F	5	−:2/5
			+: 1/5
			2+: 1/5
			3+: 1/5
LD	M	5	−:2/5
			+: 1/5
			3+: 2/5
	F	5
			−: 2/5
			+: 3/5
MD	M	5	−: 2/5*
			2+:3/5
	F	5	−: 3/5
			+: 1/5
			3+:1/5

*p<0.05 and **p<0.01vs C group with the same gender and period.

Table 5. Mean Absolute Organ Weights (g)

Group		Animal Number		Body weight		Thymus		Liver
Male
C		10		593.3±50.9		0.416±0.046		14.215±1.636
LD		10		594.2±64.3		0.375±0.098		14.185±2.172
MD		10		580.7±38.9		0.384±0.079		14.003±1.359
HD		10		563.3±30.0		0.331±0.075*		13.127±1.803
CR		5		691.4±90.5		0.385±0.066		16.408±3.552
HDR		5		618.4±64.4		0.354±0.109		14.743±2.083
Female
C		10		334.8±31.7		0.317±0.098		9.234±0.928
LD		10		321.2±32.5		0.312±0.076		8.400±0.590*
MD		10		316.7±34.4		0.318±0.109		8.666±0.791
HD		10		320.1±18.0		0.317±0.078		8.758±0.575
CR		5		334.9±29.5		0.314±0.032		8.849±1.391
HDR		5		324.9±26.2		0.299±0.060		8.593±0.824

Data showed as Mean ± SD;*p<0.05 and **p<0.01vs C group with the same gender and period; Absolute organ weights of Group C, LD, MD and HD were the end of treatment period, Group CR and HDR were the end of recovery period.

Table 6. SummaryHistopathologyChanges and Incidence Rate

 

Organs	Pathological changes	End of Treatment Period				End of Recovery Period
		C	LD	MD	HD	CR	HDR
Lung	Chronic bronchioloalveolar inflammation	2 (4/20) 3 (5/20)	—	—	1 (3/20) 2 (5/20) 3 (1/20)	1 (4/10) 2 (3/10) 3 (1/10)	1 (6/10)
Prostate	Interstitial inflammatory cell infiltration	1 (1/10) 2 (2/10)	—	—	1 (1/10) 2 (1/10)	3 (1/5)	1 (1/5)

Note: Number “1 - 4” before parentheses means the degree of histopathology changes:1-light;2-slightly;3-midrange;4-severe.

Number in the parentheses means incidence rate of histopathology changes.

“—” means no observed changes or without histopathology examination.

Conclusions:

In a reliable study conducted according to OECD 408 and in compliance with GLP, administration of the test item at a dose of 1000 mg/kg bw/day did not reveal adverse effects. Thus, the No Observed Adverse Effect Level (NOAEL) of Aluminium Magnesium Vanadium Oxide was determined to be 1000 mg/kg body weight/day for the 90-day repeated dose oral toxicity study in male and female rats.

Executive summary:

The purpose of this oral toxicity study was to assess the repeated dose toxicity of Aluminium Magnesium Vanadium Oxide, when administered daily to rats by gavage for a period of 90 days. It could provide information to determine the No-Observed Adverse Effect Level (NOAEL) and to select the dose concentrations for chronic studies.

The test item Aluminium Magnesium Vanadium Oxide was administered via gavage to groups of 10 male and 10 female Sprague Dawley rats, respectively at dose levels of 0 mg/kg bw/d (group C, corn oil only), 100 mg/kg bw/d (group LD), 300 mg/kg bw/d (group MD) and 1000 mg/kg bw/d (group HD) over a period of 90 days. To observe the reversibility or delayed occurrence of toxic effects, additional 2 satellite groups (group CR of 0 mg/kg bw/d, group HDR of 1000 mg/kg bw/d) of ten animals (five animals per sex) were scheduled for follow-up observation for a 28 days recovery period after the last administration.

During the study following observations and examinations were conducted: general observations, body weight, food consumption, functional observational battery, ophthalmologic examination, hematology, blood biochemistry and coagulation, urinalyses, organ weight, macropathology and histopathology investigations were undertaken.

Mortality

No animal died during observation period in the present study.

Clinical Signs

No test item related clinical sign was observed in any of the animals during the entire study

Body Weight

The measured mean body weight was increased with the progress of the study in all groups. There was no statistically or biologically significant effect observed on body weight in treated groups when compared with control group.

Food Consumption

The slight differences of food consumption among the groups were considered as incidental fluctuations of food intake due to the absence of dose relationship. There was no statistically or biologically significant effect observed on food consumption in treated groups during the entire study period.

Ophthalmologic Examination

No treatment related findings were observed in ophthalmologic examination in all groups at the end of treatment and recovery periods.

Functional Observational Battery

The observed changes in measured urination or defecation did not show a trend of consistency or a dose relationship. Therefore, the differences were judged to be incidental rather than treatment related.

Hematology and Blood Coagulation

Dose-dependent reduction of MPV (fL) (p<0.05) was observed in males, but there was no changes of PLT (10⁹/L) noted when compared to the control group. Dose-dependent reduction of MCV (fL) was noted in females (p<0.05), but there was no changes of RBC (10¹²/L) observed when compared to the control group. Significantly reduced means of RDW (%) (p<0.05) in females of group LD, and significantly reduced means of PDW (%) and EOS (%) (p<0.05) in females of group HD were noted, respectively. Although some of the differences attained statistical significance (p< 0.05) of the examined parameters, the values of these changes were within historical control data range.

Blood coagulation was not affected by test item in both males and females.

Clinical Biochemistry

Dose-dependent reduction of TP (g/L) (p<0.01) was observed in males when compared to the control group. Significantly elevated means of Urea (mmol/L) (p<0.05) was noted in males and females of the group HD, but there was no change of CREA (μmol/L) observed. Significantly elevated means of ALT (U/L) (p<0.05) was noted in males of group MD, and significantly reduced means of ALB (g/L) (p<0.05), AST (U/L) (p<0.05)as well as GLU (mmol/L) (p<0.01)were noted in males of group HD.

However, the values of these changes remained within the ranges of the historical control data. In addition, all clinical biochemical changes were considered lacking dose-relationship and therefore considered to be incidental rather than treatment related.

Urinalysis

Significantly changes of BIL (p<0.05) was noted in females of group LD. However, there were no significantly changes in TBIL and D-BIL in the clinical biochemistry and therefore the changes of BIL was judged to be of no toxicological significance. There were no statistically or biological significant changes compared to those in the control group in any other examination parameter of treated groups.

Pathology

All animal survived until terminal sacrifice terminal sacrifice of the exposure or recovery period. Stomach, spleen and abdominal adhesions was observed in one female (R1016) of control group. Nephrohydrosis were detected in one male (R4063) of group HD. Visible hard mass of right kidney was observed in one female (R4080) of group HD. All gross lesions noted were single observations. The macroscopic findings were judged to be incidental and not treatment related.

Organ Weight

The organ weights were recorded from the surviving animals and relative organ weights in relation to body weight were calculated. Absolute organ weights: the mean thymus gland weight of males in group HD and the mean liver weight of females in group LD was decreased (p< 0.05) when compared to group C of the same sex. The changes only were judged to be have no toxicological significance. Relative organ weights: there was no significantly change noted in any of the dosed group when compared to group C of the same sex.

Histopathology

Different degree of focal chronic inflammation observed in heart and kidney in control and HD groups. Prostate chronic inflammation was noted in few animals in control, HD and HDR group. These findings were assumed to be unrelated to treatment and judged to be spontaneous occurring background pathology findings in this strain of this age. At the end of treatment period, local proliferation and focal inflammation lesions focal inflammation in kidney in individual animal was judged to be incidental rather than treatment-related. No treatment related microscopic findings were observed in kidney of other animals of all groups at the end of treatment and recovery periods. There were no test item treated related histopathological changes found in the 90-day repeated dose oral toxicity with aluminium magnesium vanadium oxide.

Discussion

No treatment related findings were observed in all groups at the end of treatment and recovery periods, including mortality, clinical signs, body weight, food consumption, ophthalmologic examination, functional observational battery, hematology and blood coagulation, clinical biochemistry, urinalysis, pathology and organ weight. Different degree of focal chronic inflammation observed in heart and kidney in control and HD groups. Prostate chronic inflammation was noted in few animals in control, HD and HDR group. These findings were assumed to be unrelated to treatment and judged to be spontaneous occurring background pathology findings in this strain of this age. At the end of treatment period, local proliferation and focal inflammation lesions focal inflammation in kidney in individual animal was judged to be incidental rather than treatment-related. No treatment related microscopic findings were observed in kidney of other animals of all groups at the end of treatment and recovery periods. There were no test item treated related histopathological changes found in the 90-day repeated dose oral toxicity with aluminium magnesium vanadium oxide.

Conclusion

Based on the data generated in this study, the NOAEL (No Observed Adverse Effect Level) of Aluminium Magnesium Vanadium Oxide is considered to be 1000 mg/kg body weight/day for the 90-Day repeated dose oral toxicity study in male and female rats.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct 2014 - 16 Mar 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

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)

Remarks:

National(CFDA) GLP certificate

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Center of Academy of Military Medical Sciences
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) x 10-11wks
- Weight at study initiation: (P) Males: 317.9-359.8 g; Females: 218.7-270.1 g
- Fasting period before study: No
- Housing:The rats were housed five of one sex per cage at treatment before mating and after mating (only males). The pregnant animals were single-housed after mated. Animals were mated in the ratio of 1:1 (Male to Female) during the mating period.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40-70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light):12 hrs dark/12 hrs light
IN-LIFE DATES: from 22 Dec 2014 to 13 Feb 2015

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test item formulations were freshly prepared every day immediately prior to application.
Appropriate amounts of the test item were weighted into a tared glass vial on a suitable precision balance and the vehicle (corn oil) was added to obtain the designed final concentration of the test item formulations. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before administration. The vehicle (corn oil) was also used as the control item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil is frequently used as a suitable vehicle in animal experiment when the solubility of test article is low in water. Moreover, corn oil has been proposed as a suitable vehicle by the OECD (OECD Guidelines for Testing of Chemicals 421Reproduction/Developmental Toxicity Screening Test (Adopted 24, July, 1995). Sponsor has agreed to use corn oil as vehicle in this study.
- Concentration in vehicle: Not specified
- Amount of vehicle (if gavage): 0.4 mL /100g body weight
- Lot/batch no. (if required): QS130302011011
- Purity: according to the nactional guidance on maize oil (GB 19111-2003)

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): The pregnant animals were single-housed after mated
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

no

Remarks:

No analytical need for poor soluble inorganic substance

Duration of treatment / exposure:

approximately 54 days for female (day14 pre-mating to day 4 post-partum )
28 days for male (day14 pre-mating and day14 of mating )

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose (LD) group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

medium dose (MD) group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose(HD) group

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Toxicological Information: LC50 (inhalation) > 5 mg/L; LD50 (dermal) > 2000 mg/kg body weight; LD50 (oral) > 2000 mg/kg body weight; no signs of toxicity at acute sublethal concentration;

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Cage side observations included: mortality/viability

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed at receiving, grouping, first day of dosing and weekly thereafter, and at termination. During pregnancy, females were weighed every 3 days and within 24 hours after parturition (day 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities;

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed at day 28 of dosing
- Maternal animals: Pregnant females animals were killed at day 4 post-partum, and non-pregnant females were killed 24-26 days after the last day of the mating period.

GROSS NECROPSY
- At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. The pregnancy status of the females was ascertained. The number of implantation sites, the counting of corpora lutea, and the number of absorbed fetus and dead fetus were recorded. Uteri that appear non-gravid should be further examined by 8% ammonium sulphide staining for 20 min to confirm the non-pregnant status with or without implantation.

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination was performed on the ovaries, uterus, testes and epididymides of the animals of the highest dose group and the control group. Examinations were not extended to the animals of other dosage groups, because no changes were seen in the highest dose group. The testes and epididymides of male adult and ovaries and uterus of female adult animals were weighed. The absolute organ weights and the relative organ weights on the basis of the body weight were calculated. The ovaries, uterus, testes, epididymides, accessory sex organs and all organs showing macroscopic lesions of all adult animals were preserved using Bouin’s fixative.

Postmortem examinations (offspring):

SACRIFICE
Dead pups and pups killed at day 4 post-partum were examined externally for gross abnormalities.

Statistics:

The numerical data obtained in body weight, food consumption, organ weight and relative organ weight were subjected to multiple comparison tests for statistical significance of difference. The data were analyzed for homogeneity of variance and the homogeneous data were subjected to one-way analysis of variance. If significant differences were observed among groups, group mean difference between the control group and the dose group was subjected to multiple comparison test by the Dunnet t test (for homogeneous data). The enumeration data were analyzed using chi-square Test. These statistics were performed with SPSS 18.0 software (p<0.05 was considered as statistically significant)
Individual animal data were provided. Additionally, all data were summarized in tabular form, showing for each test group the number of animals at the start of the test, the number of animals found dead during the test or killed for human reasons, the time of any death or human kill, the number of fertile animals, the number of pregnant females, the number of animals showing signs of toxicity, a description of the signs of toxicity observed, including time of onset, duration, and severity of any toxic effects, the types of histopathological changes, and all relevant litter data.

Reproductive indices:

Copulation index (%) = (numbers of animals mated/number of animals paired) × 100%
Fertility index (%) = (numbers of pregant female animals/number of animals paired) × 100%
Gestation index (%) = (numbers of pregnant female animals with live pups born/number of pregnant animals) × 100%
Viability index (%) = (numbers of pups alive Day 4 of age/numbe of pups born alive) × 100%

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

No mortalities were observed in pregnant animals during the entire period of the study. All the parental animals mated successfully, and gave birth within 22 days of pregnancy. The number of animals with pregnancy period ≤21 days in LD group was significant reduced when compared to the control group (P<0.05), while no significant changes were noted in MD and HD groups (P>0.05). There were no significant changes observed in dams with live young born, dams with live young born at day 4 post-partum, number of corpora lutea/dam (mean) and number of implants/dam (mean) in treatment groups when compared to control group. No statistically significant difference was found in reproductive indices in treatment groups when compared to control group.

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 adverse effects were observed

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No test item related clinical signs were observed in any pups during the 4 days post-partum.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

For pups, 16 pups were born dead (R1014-F13 (control group), R2038-F19, R2042-F16 (low dose group), R3061-M7, R3064- M9, R3066-M6, R3071-M8, R3071-M9, R3071-M10, R3071-M11, R3071-F17 (intermediate dose group) and R4090-M5, R4090-F12, R4090-F13, R4090-F14, R4091-F15 (high dose group)) and 2 pups were dead at day 4 post-partum (R1018-F13 and R2044-M1). There were no significant differences in treatment groups were found in dead pups at birth when compared with control group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with control group, no statistically significant differences were found in litter weight at birth (mean) (P>0.05) and litter weight at day 4 (mean) (P>0.05). There were significant reduction in pup weight at birth (mean), pup weight at day 4 (mean), in LD group when compared to control group, while no significant changes were found in MD and HD group. The significantly pup weight changes only noted in LD group was judged to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal pups observed (P>0.05).

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

When compared with control group, no statistically significant differences were found in live/dead pups/dam at birth (P>0.05), live/dead pups/dam at day 4 (P>0.05), sex ratio (m/f) at birth (P>0.05), sex ratio (m/f) at day 4 (P>0.05), abnormal pups (P>0.05), loss of offspring (pre-implantation, pre-natal/post-implantations, post-natal). There were significant reduction in pup body length at birth (mean) and pup tail length at birth (mean) in LD group when compared to control group, while no significant changes were found in MD and HD group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed

Key result

Reproductive effects observed:

no

Results:

Table 1. Mean Relative Organ Weights to Body Weight

Group	Sex	n	Testes/ Uterus
			Left	Right
C	M	12	0.340±0.026	0.338±0.030
	F	9	0.232±0.042
LD	M	12	0.352±0.049	0.356±0.055
	F	11	0.276±0.043*
MD	M	12	0.340±0.039	0.354±0.034
	F	10	0.265±0.035
HD	M	12	0.375±0.039*	0.376±0.040*
	F	11	0.246±0.043

Data showed as Mean ± SD.

 

Table 2. Tabulated summary report of effects on reproduction/development

Observations		Values
Dosage (mg/kg)		0		100		300		1000
Females achieving pregnancy (N)		9		11		10		11
Conceiving days 1 - 5 (N)		9		11		10		11
Conceiving days 6 -14 (N)		0		0		0		0
Pregnancy ≤21 days (N)		5		11*		8		4
Pregnancy = 22 days (N)		4		0		2		7
Pregnancy≥23 days (N)		0		0		0		0
Pup weight at birth (mean±SD)		7.17±0.76		6.83±0.67*		7.19±0.62		7.09±0.70
Pup weight at day 4 (mean±SD)		9.76±1.42		8.86±1.26*		9.46±1.17		9.89±1.36
Pup body length at birth (mean±SD)		51.65±1.51		51.07±1.92*		51.76±1.17		51.88± 1.64
Pup tail length at birth (mean±SD)		16.96±1.53		16.56±1.23*		16.76±0.84		16.71± 1.28

* P<0.05, ** P<0.01

Conclusions:

In a reliable study conducted according to OECD 421 and in compliance with GLP, administration of the test item at a dose of 1000 mg/kg bw/day did not reveal adverse effects. Thus, the No Observed Adverse Effect Level (NOAEL) of Aluminium Magnesium Vanadium Oxide was determined to be 1000 mg/kg body weight/day for reproduction/developmental toxicity screening in males and females.

Executive summary:

Objective

The objective of this study was to assess the possible effects of the test item Aluminium Magnesium Vanadium Oxide on male, female fertility and embryofetal development in Sprague Dawley rats when administrated orally.

Study Design

The test item Aluminium Magnesium Vanadium Oxide was administered via gavage to groups of 5 male and 5 female Sprague Dawley rats, respectively at dose levels of 0 mg/kg bw/d (group C, corn oil only), 100 mg/kg bw/d (group LD), 300 mg/kg bw/d (group MD) and 1000 mg/kg bw/d (group HD). The test item was administrated daily during 14 days pre mating period and 14 days mating period in both male and female, during gestation period and up to post natal day 4 in females. Animals were mated in the ratio of 1:1(Male to female). The non-pregnant females were still dosed to the day 24~26 post-mating.

During the period of administration, clinical signs, body weights, food consumption, fertility and mating performance, organ weights, and microscopic examinations were recorded for all parental animals. Litter size, litter observation, body weights, length and abnormalities were recorded for the offspring.

Results

No animal died and no test item related clinical signs were observed during the study. No statistically or toxicological significant effect was observed on body weight, food consumption, organ weights, histopathology findings, mating performanceand fertility, pregnancy and litter parameters in the study.

Conclusion

Based on the data generated from this study, the No Observed Adverse Effect Level (NOAEL) of Aluminium Magnesium Vanadium Oxide is 1000 mg/kg body weight/day for reproduction/developmental toxicity screening in males and females.

Materials and methods

Test animals

Species:

rat