Dizinc orthosilicate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is conducted according to OECD TG 423 with some modifications in terms of different dose levels, usage of sexes, animal number, and inclusion of hematology, biochemical parameters and histopathology evaluation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Cross-reference 2

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is conducted according to OECD TG 423 with some modifications in terms of different dose levels, usage of sexes, animal number, and inclusion of hematology, biochemical parameters and histopathology evaluation.

Justification for type of information:

See attached justification in IUCLID section 13.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

there were modifications in terms of different dose levels, usage of sexes, animal number, and inclusion of hematology, biochemical parameters and histopathology evaluation.

GLP compliance:

not specified

Remarks:

the publication does not specify GLP compliance

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and housing conditions:
- Experimental animals were obtained from in-house animal facility. All procedures using animals were reviewed and approved by the institutional animal ethics committee.
- The healthy Sprague Dawley rats of both sex, aged between 8 and 9 weeks and body weights of 180–220 g were used. Females were nulliparous and nonpregnant.
- Animals were housed in polypropylene cages with stainless steel grills and gamma-irradiated corn cobs were used as bedding. Bedding material, cages, grills and water bottles were changed on alternate days.
- Animals were housed individually sex wise and group wise.
- Animals were acclimated for a minimum period of 5 d in the controlled environment (temperature: 22 + 3C; relative humidity: 50 + 20% and light: 12-h light/dark cycle) and ad libitum supply of reverse osmosis water and a standard rodent pellet feed. Feed alone was withdrawn overnight prior to the dosing and following dosing, for a period of 3 h.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

- The test substance was suspended in distilled water and administered through oral gavage once at dose levels of 5, 50, 300, 1,000 and 2,000 mg/kg bw.
- The test solution was prepared shortly prior to the administration. The dose volume maintained for all the groups was maximum (10 mL/kg bw).
Similarly, control group of animals (5 males and 5 females) were dosed with distilled water alone.

Doses:

0, 5, 50, 300, 1,000 and 2,000 mg/kg bw

No. of animals per sex per dose:

Five/sex/dose

Control animals:

yes

Details on study design:

- Animals were observed for mortality/morbidity, clinical signs of toxicity, weekly body weight and weekly food consumption during the experimental period.
- At the end of 14 d of administration, the animals were killed and the blood was obtained through ophthalmic vein. The organs such as esophagus,
stomach, small and large intestines, liver, spleen, thymus, mandibular and mesenteric lymphnodes, kidney, urinary bladder, heart, pancreas, brain, lungs ovaries and testes were collected, and all the organs were kept in 10% buffered formalin and the testes in modified Davidson fluid.


METHOD DETAILS
- Clinical biochemistry: The serum obtained after centrifuging was analyzed for biochemical parameters such as creatinine, albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), amylase, aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium, cholinesterase, total cholesterol, glucose, HDL cholesterol, iron, phosphorus, total protein, triglycerides, urea and zinc using Humastar 300 fully automated biochemistry analyzer (Human GmbH., Germany), and sodium, potassium and chlorides in serum were analyzed on Day 14 by means of a humalyte electrolyte analyzer (Human GmbH., Germany).
- Hematology: Blood treated with EDTA was used for analyzing hematology parameters such as erythrocyte count (red blood cell [RBC]), hemoglobin, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) count, total leucocyte count (white blood cell [WBC]) and differential count (five parameters namely neutrophils, eosinophils, basophils, lymphocytes and monocytes)were determined on Day 14 using Bayer ADVIA 120, fully automated hematology analyzer. Clotting time of blood was determined by capillary tube method.
- Necropsy: Gross pathology was performed at the end of experimental period (Day 14).
- Histopathology: Histopathology of organs (liver, spleen, kidneys, heart, adrenals, lungs, pancreas, stomach, esophagus, small and large intestine) was evaluated. Tissues were collected and preserved in 10% buffered formalin. All tissues required for histopathology evaluation were subjected to dehydration procedure and processed in tissue processor, embedded in paraffin wax and prepared sections of 5–8 mm thickness and stained with hematoxylin-eosin stain.

Statistics:

- The data was expressed as mean +/- standard deviation for statistical analysis. A comparison of treated rats with control groups was done using Newman–Keuls multiple comparison test. The data found to be heterogeneous were subjected to nonparametric-Kruskal–Wallis multiple comparison Z value test.
- The alpha level at which all tests were conducted is 0.05, and the NCSS 2007 software was used for analysis.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Not reported in the article, but can be inferred based on the available study description.

Mortality:

No details available.

Clinical signs:

other: No details available

Gross pathology:

No gross pathological lesions were observed in any of the treatment groups.

Other findings:

Clinical biochemistry (see Table 1 for details):
- The results indicated that the plasma ALT and AST were significantly higher than the controls in both the sexes.
- There is an inverse dose-dependent increase in the AST and ALT serum levels in animals treated with nano-size ZnO.
- Calcium levels in the serum were also significantly high from control. However, there are no significant differences in rest of the parameters analyzed.

Hematology:
- There were no statistically significant changes in the hematologic parameters when compared to control.

Histopathology:
Animals treated with nano-size ZnO showed lesions in liver and pancreas. In addition to this, animals treated with nano-size ZnO at 2,000, 1,000, 300, 50 and 5 mg/kg bw showed lesions in stomach and heart as well.

Table1: Clinical biochemistry parameters (males and females)

Groups	ALT	AST	Ca
Males
G1 (N-5)	747.0 ± 120.6*	687.4 ± 83.1*	10.1 ± 0.1*
G2 (N-50)	514 ± 88.0*	553.6 ± 55.5*	10.3 ± 0.2*
G3(N-300)	428.6 ± 111.5*	509.6 ± 41.5*	10.6 ± 0.4*
G4 (N-1000)	395.2 ± 57.8*	411.8 ± 66.6*	10.2 ± 0.2*
G5(N-2000)	298.4 ± 119.1*	357.4 ± 69.3*	10.1 ± 0.1*
Females
G1 (N-5)	712.2 ± 169.5*	501.6 ± 49.3*	10.0 ± 0.3
G2 (N-50)	521.4 ± 39.8*	404.8 ± 86.7*	9.6 ± 0.8
G3(N-300)	464.2 ± 61.2*	345.8 ± 50.1	9.6 ± 0.7
G4 (N-1000)	355.6 ± 106.6*	276.6 ± 47.6*	10.2 ± 0.8
G5(N-2000)	248.6 ± 126.4*	226.8 ± 18.8*	10.0 ± 0.4

ALP: alkaline phosphatase, ALT: alanine transaminase, AST: aspartate transaminase, Ca: calcium.

*Statistically different from control; p <0.005 (n=5)

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Although an LD50 value has not been reported in the study report, but based on the study description, the LD50 value can be established at >2,000 mg/kg bw.

Executive summary:

An acute oral toxicity study was performed with ZnO nanoparticles using Sprague Dawley rats according to OECD guideline 423. A group of 10 fasted animals (five males and five females) were administered with 5, 50, 300, 1,000 and 2,000 mg/kg bw of ZnO nanoparticles suspended in distilled water once through oral gavage. The animals were observed for mortality/morbidity, clinical signs of toxicity, weekly body weight and weekly food consumption during the experimental period. The study focused on the effects of the test substance on biochemical and hematological parameters which were analyzed on Day 14 of administration. At the end of 14 days the animals were sacrificed and subjected to gross pathological examination along with histopathology or organs.

An inverse dose-dependent increase was noted in AST, ALT serum levels when compared with control. Calcium levels in the serum were also significantly high from control. However, there are no significant differences in rest of the parameters analyzed. There were also no statistically significant changes in the hematologic parameters. No gross pathological lesions were observed in any of the treatment groups. Histopathological evaluation showed incidences of microscopic lesions in liver, pancreas, heart and stomach which were higher at lower doses compared to higher dose.

Although an LD50 value was not reported in the study report, based on the study description and considering that no mortality occurred at any of the tested dose levels, the LD50 value can be established at >2,000 mg/kg bw.

Overall the authors concluded that the high toxicity at low doses which were evident in the present study may be due to the fewer number of nanoparticles in that mass, which may result in less agglomeration thereby making them penetrate into the cells.

Data source

Reference

Reference Type:

publication

Title:

Toxicity of zinc oxide nanoparticles through oral route.

Author:

Pasupuleti S, Alapati S, Ganapathy S, Anumolu G, Pully, NR and Prakhya BM

Year:

2012

Bibliographic source:

Toxicol. Ind. Health, 28(8):675–686

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

there were modifications in terms of different dose levels, usage of sexes, animal number, and inclusion of hematology, biochemical parameters and histopathology evaluation.

GLP compliance:

not specified

Remarks:

the publication does not specify GLP compliance

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Zinc oxide

EC Number:

215-222-5

EC Name:

Zinc oxide

Cas Number:

1314-13-2

Molecular formula:

OZn

IUPAC Name:

oxozinc

Test material form:

solid: nanoform

Details on test material:

- Name of test material (as cited in study report): ZnO nanoparticles
- Stock No. 5810HT
- Nanoparticle synthesis: ZnO nanoparticles were synthesized by wet chemistry method.
- Physicochemical characterization of ZnO nanoparticles:
- Size of nanoparticle: The average size of the nano ZnO is 63 nm determined in SEM analysis; the average size of nano ZnO (in solution) was 224.7 nm determined using dynamic light scattering (DLS).
- Polydispersity index: 0.305
- Surface area: 50 m2/g determined using BET (Brunauer, Emmett, Teller) analysis
- Zeta potential: The zeta potential of nano-size ZnO was -30.9 mV determined using Zetasizer.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and housing conditions:
- Experimental animals were obtained from in-house animal facility. All procedures using animals were reviewed and approved by the institutional animal ethics committee.
- The healthy Sprague Dawley rats of both sex, aged between 8 and 9 weeks and body weights of 180–220 g were used. Females were nulliparous and nonpregnant.
- Animals were housed in polypropylene cages with stainless steel grills and gamma-irradiated corn cobs were used as bedding. Bedding material, cages, grills and water bottles were changed on alternate days.
- Animals were housed individually sex wise and group wise.
- Animals were acclimated for a minimum period of 5 d in the controlled environment (temperature: 22 + 3C; relative humidity: 50 + 20% and light: 12-h light/dark cycle) and ad libitum supply of reverse osmosis water and a standard rodent pellet feed. Feed alone was withdrawn overnight prior to the dosing and following dosing, for a period of 3 h.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

- The test substance was suspended in distilled water and administered through oral gavage once at dose levels of 5, 50, 300, 1,000 and 2,000 mg/kg bw.
- The test solution was prepared shortly prior to the administration. The dose volume maintained for all the groups was maximum (10 mL/kg bw).
Similarly, control group of animals (5 males and 5 females) were dosed with distilled water alone.

Doses:

0, 5, 50, 300, 1,000 and 2,000 mg/kg bw

No. of animals per sex per dose:

Five/sex/dose

Control animals:

yes

Details on study design:

- Animals were observed for mortality/morbidity, clinical signs of toxicity, weekly body weight and weekly food consumption during the experimental period.
- At the end of 14 d of administration, the animals were killed and the blood was obtained through ophthalmic vein. The organs such as esophagus,
stomach, small and large intestines, liver, spleen, thymus, mandibular and mesenteric lymphnodes, kidney, urinary bladder, heart, pancreas, brain, lungs ovaries and testes were collected, and all the organs were kept in 10% buffered formalin and the testes in modified Davidson fluid.


METHOD DETAILS
- Clinical biochemistry: The serum obtained after centrifuging was analyzed for biochemical parameters such as creatinine, albumin, alkaline phosphatase (ALP), alanine aminotransferase (ALT), amylase, aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium, cholinesterase, total cholesterol, glucose, HDL cholesterol, iron, phosphorus, total protein, triglycerides, urea and zinc using Humastar 300 fully automated biochemistry analyzer (Human GmbH., Germany), and sodium, potassium and chlorides in serum were analyzed on Day 14 by means of a humalyte electrolyte analyzer (Human GmbH., Germany).
- Hematology: Blood treated with EDTA was used for analyzing hematology parameters such as erythrocyte count (red blood cell [RBC]), hemoglobin, hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) count, total leucocyte count (white blood cell [WBC]) and differential count (five parameters namely neutrophils, eosinophils, basophils, lymphocytes and monocytes)were determined on Day 14 using Bayer ADVIA 120, fully automated hematology analyzer. Clotting time of blood was determined by capillary tube method.
- Necropsy: Gross pathology was performed at the end of experimental period (Day 14).
- Histopathology: Histopathology of organs (liver, spleen, kidneys, heart, adrenals, lungs, pancreas, stomach, esophagus, small and large intestine) was evaluated. Tissues were collected and preserved in 10% buffered formalin. All tissues required for histopathology evaluation were subjected to dehydration procedure and processed in tissue processor, embedded in paraffin wax and prepared sections of 5–8 mm thickness and stained with hematoxylin-eosin stain.

Statistics:

- The data was expressed as mean +/- standard deviation for statistical analysis. A comparison of treated rats with control groups was done using Newman–Keuls multiple comparison test. The data found to be heterogeneous were subjected to nonparametric-Kruskal–Wallis multiple comparison Z value test.
- The alpha level at which all tests were conducted is 0.05, and the NCSS 2007 software was used for analysis.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Not reported in the article, but can be inferred based on the available study description.

Mortality:

No details available.

Clinical signs:

other: No details available

Gross pathology:

No gross pathological lesions were observed in any of the treatment groups.

Other findings:

Clinical biochemistry (see Table 1 for details):
- The results indicated that the plasma ALT and AST were significantly higher than the controls in both the sexes.
- There is an inverse dose-dependent increase in the AST and ALT serum levels in animals treated with nano-size ZnO.
- Calcium levels in the serum were also significantly high from control. However, there are no significant differences in rest of the parameters analyzed.

Hematology:
- There were no statistically significant changes in the hematologic parameters when compared to control.

Histopathology:
Animals treated with nano-size ZnO showed lesions in liver and pancreas. In addition to this, animals treated with nano-size ZnO at 2,000, 1,000, 300, 50 and 5 mg/kg bw showed lesions in stomach and heart as well.

Any other information on results incl. tables

Table1: Clinical biochemistry parameters (males and females)

Groups	ALT	AST	Ca
Males
G1 (N-5)	747.0 ± 120.6*	687.4 ± 83.1*	10.1 ± 0.1*
G2 (N-50)	514 ± 88.0*	553.6 ± 55.5*	10.3 ± 0.2*
G3(N-300)	428.6 ± 111.5*	509.6 ± 41.5*	10.6 ± 0.4*
G4 (N-1000)	395.2 ± 57.8*	411.8 ± 66.6*	10.2 ± 0.2*
G5(N-2000)	298.4 ± 119.1*	357.4 ± 69.3*	10.1 ± 0.1*
Females
G1 (N-5)	712.2 ± 169.5*	501.6 ± 49.3*	10.0 ± 0.3
G2 (N-50)	521.4 ± 39.8*	404.8 ± 86.7*	9.6 ± 0.8
G3(N-300)	464.2 ± 61.2*	345.8 ± 50.1	9.6 ± 0.7
G4 (N-1000)	355.6 ± 106.6*	276.6 ± 47.6*	10.2 ± 0.8
G5(N-2000)	248.6 ± 126.4*	226.8 ± 18.8*	10.0 ± 0.4

ALP: alkaline phosphatase, ALT: alanine transaminase, AST: aspartate transaminase, Ca: calcium.

*Statistically different from control; p <0.005 (n=5)

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Although an LD50 value has not been reported in the study report, but based on the study description, the LD50 value can be established at >2,000 mg/kg bw.

Executive summary:

An acute oral toxicity study was performed with ZnO nanoparticles using Sprague Dawley rats according to OECD guideline 423. A group of 10 fasted animals (five males and five females) were administered with 5, 50, 300, 1,000 and 2,000 mg/kg bw of ZnO nanoparticles suspended in distilled water once through oral gavage. The animals were observed for mortality/morbidity, clinical signs of toxicity, weekly body weight and weekly food consumption during the experimental period. The study focused on the effects of the test substance on biochemical and hematological parameters which were analyzed on Day 14 of administration. At the end of 14 days the animals were sacrificed and subjected to gross pathological examination along with histopathology or organs.

An inverse dose-dependent increase was noted in AST, ALT serum levels when compared with control. Calcium levels in the serum were also significantly high from control. However, there are no significant differences in rest of the parameters analyzed. There were also no statistically significant changes in the hematologic parameters. No gross pathological lesions were observed in any of the treatment groups. Histopathological evaluation showed incidences of microscopic lesions in liver, pancreas, heart and stomach which were higher at lower doses compared to higher dose.

Although an LD50 value was not reported in the study report, based on the study description and considering that no mortality occurred at any of the tested dose levels, the LD50 value can be established at >2,000 mg/kg bw.

Overall the authors concluded that the high toxicity at low doses which were evident in the present study may be due to the fewer number of nanoparticles in that mass, which may result in less agglomeration thereby making them penetrate into the cells.