Reaction products of sodium glucoheptonate with zinc sulfate and sodium hydroxide

Endpoint summary

Description of key information

oral

In an oral toxicity study with the registered substance (OECD 423), two sets of fasted Wistar rats (3 femals/set) (10 - 11 weeks) were given a single oral dose of 2000 mg test material /kg bw and observed for 14 days. The conducted study was in compliance with GLP (GLP certificate issued by the Government of India, Department of Science and Technolog; National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA)).There were no treatment-related mortality, clinical signs and changes in body weight or necropsy findings observed so the LD50 is >2000 mg/kg bw. Based on the results of this study, the indication of the classification of the registered substance is as follows: EU GHS (CLP) criteria not met.

inhalation

To address the endpoint acute toxicity (inhalation) read across on zinc oxide nanoparticles was performed within the frame of a weight of evidence approach.The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

The available studies indicate certain inhalatory toxicity of zinc, when applied as nano- or small microparticle. When evaluating these results, it has to be considered that the toxicity of nanoparticles arise rather from their increased reactivity due to their high surface to mass ratio than from their chemical constitution. This is not relevant for zinc glucoheptonate, which has no particles < 40 µm. In addition, due to the particle size of > 40 µm zinc glucoheptonate particles are not able to reach the alveolar region. Another property limiting any inhalation toxicology of zinc glucoheptonate is the fact that the toxicologically relevant zinc ion is complexed by a sugar residue with a high molecular weight in the target substance zinc glucoheptonate. Therefore, an enormous amount of the substance would have to be inhaled to reach a zinc concentration in the lungs being toxically relevant. Therefore no hazard via inhalation is expected for zinc glucoheptonate.

Zinc oxide

The predominant toxic effect of inhaled ZnO is metal fume fever. The term metal fume fever describes a variety of symptoms, including fever, chills, dyspnea, muscle soreness, nausea, and fatigue, which occur in workers occupationally exposed to airborne metal oxide particles.

The U.S. Occupational Safety and Health Administration (as matters stand 2005) permissible exposure limit for zinc oxide is (TLV = 5.0 mg/m3) inspired air, fume or respirable dust, as an 8-hour time-weighed average, over a 40-hour working week.

Wang et al., 2010 studied the toxic effect of ZnO nanoparticles. Male Wistar rats were consecutively treated with ZnO nanoparticles (particle size of 20 nm) at 2.5 mg/kg body weight, twice daily tor 3 days. The ZnO nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer.

Once ZnO nanoparticles entered the body via inhalation, it became systemically available and caused toxic effects in internal organs other than in the lungs. Pulmonary retention, extrapulmonary translocation, and redistribution were considered to be the essential mechanisms of organ damage induced by inhaled nanoparticles. Obvious lesions of liver and lung were induced and the levels of serum ALT, AST, ALP, CK, and LDH were all significantly decreased compared with the control groups (Wang et al. 2010).

Gordon et al. (1992) examined the time course and dose-response of the pulmonary injury produced by inhaled ZnO in guinea pigs, rats and rabbits. The test animals were exposed to 0, 2.5, or 5.0 mg/m3 ZnO with a particle size of 0.17 µm for up to 3 h. After that their lungs were lavaged. Both the lavage fluid and recovered cells were examined for evidence of inflammation or altered cell function. The lavage fluid from guinea pigs and rats exposed to 5 mg/m3 had significant increases in total cells, lactate dehydrogenase, b-glucuronidase, and protein content. These changes were greatest 24 h after exposure. Guinea pig alveolar macrophage function was depressed as evidenced by in vitro phagocytosis of opsonized latex beads. Significant changes in lavage fluid parameters were also observed in guinea pigs and rats exposed to 25 mg/m3 ZnO. In contrast, rabbits showed no increase in biochemical or cellular parameters fallowing a 2-h exposure to 5 mg/m3 ZnO. Differences in total lung burden of ZnO, as determined in additional animals by atomic absorption spectroscopy, appeared to account for the observed differences in species responses. Although the lungs of guinea pigs and rats retained approximately 20% and 12% of the inhaled dose, respectively, rabbits retained only 5%. Together, these studies demonstrate that a single 2- to 3-h exposure to ZnO at the current threshold limit value (TLV) of 5 mg/m3 does elicit adverse health effects in several mammalian species. Pulmonary effects in rats and guinea pigs were also observed at a concentration of 2.5 mg/m3.

human data

Gordon et al. (1992) also studied inhalation toxicity of ZnO (particle size of 0.17 µm) in 4 human volunteers (reference in Exposure related observations in humans: other data). The human volunteers were exposed to 5 mg/m3 zinc oxide. Each of the subjects responded with one or more of the classic symptoms of metal fume fever approximately 6 to 10 h after a 2-h exposure to 5 mg/m3 ZnO, although no changes in pulmonary function were observed. This shows that a single 2- to 3-h exposure to ZnO at the current threshold limit value (TLV) of 5 mg/m3 does elicit adverse health effects in human.

Beckett et al. (2005) (reference in section Exposure related observations in humans: other data) studied the inhalation toxicity of fine (0.1 - 1 µm diameter) and ultrafine fractions (< 0.1 µm diameter) of freshly generated zinc oxide in healthy humans. The test subjects were exposed to a concentration of 0.5 mg/m3 of the fine or ultrafine fraction for 2 hours at rest. Preexposure and follow-up studies of symptoms, leukocyte surface markers, hemostasis, and cardiac electrophysiology were conducted to 24 hours post-exposure. Induced sputum was sampled 24 hours after exposure. No differences were detected between any of the three exposure conditions at this level of exposure. Freshly generated zinc oxide in the fine or ultrafine fractions inhaled by healthy subjects at rest at a concentration of 0.5 mg/m3 for 2 hours is below the threshold for acute systemic effects as detected by these endpoints. The tested dose was too low a dose to show any effect, or a “no observable effect level” of exposure.

In these studies effects were observed with zinc oxide particles having diameters between 20 nm and 170 nm as test substance.The majority of the particles of zinc glucoheptonate (87.1 %), on the other hand, has a size between 100 and 800 µm. 13.9 % of the particles are smaller than 100 µm but bigger than 40 µm. There are no particles smaller than 40 µm. Therefore an extrapolation from the toxicity of the zinc nano- or small microparticles to the toxic effect of the chelated zinc ions of zinc glucoheptonate is difficult. Nanoparticles have a special toxicological behaviour. Because of the high surface to mass ratio, nanoparticles are more reactive than larger-sized particles of the same chemistry (Oberdörster et al., 2005). In addition, other absorption routes are accessible for nanoparticles. When inhaled, they are efficiently deposited in all regions of the respiratory tract; they evade specific defense mechanisms; and they can translocate out of the respiratory tract via different pathways and mechanisms (endocytosis and transcytosis) (Oberdörster et al., 2005).

So, the toxicological profile of zinc glucoheptonate will differ from the one of ZnO nanoparticles. The inhalatary absorption rate is expected to be markedly reduced when compared to ZnO nanoparticles. This is due to the occurence of zinc complexed by a sugar residue with a high molecular weight in the target substance and the fact that zinc glucoheptonate has no fractions > 40 µm being able to reach the alveolar region. In addition, the high reactivity up to the increased surface to mass ratio is not relevant for zinc glucoheptonate. Thus, zinc oxide nanoparticles are expected to be much more toxic than zinc glucoheptonate.

 

dermal route

The acute dermal toxicity endpoint is waived since there is no acute hazard for Zn glucoheptonate expected via dermal route of exposure. No hazard can be attributed to glucoheptonate moiety. Glucoheptonate is a sugar anologue and was assessed as safe for veterinary and medicinal products (EMEA, 1998). Glucoheptonate is used as imaging agent in patients with brain, renal and pulmonal tumors (Waxman et al. 1975, Boyd et al. 1973, Passamonte et al. 1983). No acute dermal hazard can be attributed to zinc cation (WHO 2001, Van Huygevoort (1999c), cited in EUR 21170 EN (RAR Zinc sulphate) 2008). Zinc favours wound healing and is therefore used in a number of medicinal dermal applications (Kaufmann et al. 2013, Gordon 1981, cited in ATSDR 2005). Moreover, absorption of Zn glucoheptonate in its chelated form and as Zn cation and glucoheptonate anion (very soluble substance, Water solubility of 500-550 g/L, logPow of -6.58) will be extremely low via the skin, no significant amounts of Zn or glucoheptonate can be absorbed into systemic circulation. Therefore, the systemic doses at which adverse effects would occur after oral or iv administration, can never be reached by dermal route. Based on these considerations, dermal acute toxicity study is not necessary.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.06.2016 - 12.07.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

issued by the Government of India, Department of Science and Technolog; National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed in a cool and well ventilated place.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 170.2 -196.2 g
- Fasting period before study: Rats were fasted overnight prior to dosing until three hours post-dosing.
- Housing: 3 rats per cage. Polypropylene rat cages covered with stainless steel grid top were used. Autoclaved clean rice husk was used as the bedding material.
- Diet: ad libitum (Teklad Certified Global HIgh Fiber Rat/Mice Feed (Envigo, USA))
- Water: ad libitum (UV sterilized water filtered through Reverse Osmosis water filtration system)
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23 °C
- Humidity (%): 57 - 67 %
- Air changes (per hr): minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Reverse Osmosis (RO) Water

Details on oral exposure:

The actual dose formulation was prepared using RO water as vehicle. Required quantity were mixed in RO water and the final volume was made up to 10 mL. Gavage solution were prepared freshly prior to dosing on all the occasions.
Individual dose volume was adjusted according to body weight. All rats were dosed by oral gavage (day 0) using a BD 1 mL disposable syringe. Rats were fasted overnight prior to dosing until three hours post-dosing.

Doses:

2000 mg/ kg bw

No. of animals per sex per dose:

Two sets of 3 female rats, each.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Rats were observed for signs of toxicity and mortality at 0.5, 1, 2, 3, 4 and 6 hours post-administration on the day of dosing. Subsequently, rats were observed twice a day for morbidity and mortality for 14 days following oral dosing. The clinical signs were recorded once a day. Individual body weight was recorded prior to dosing on day 0 and on days 7 and 14.
- Necropsy of survivors performed: yes. All rats were euthanised by carbon dioxide asphyxiation.
- Other examinations performed: gross pathology examiation, consisting of external examination and opnening of the abdominal and thoracic cavities.

Statistics:

not reported

Preliminary study:

not applicable

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed in rats treated with 2000 mg test material /kg bw.

Clinical signs:

other: No clinical sign was observed in all rats treated with 2000 mg test material /kg bw.

Gross pathology:

In absence of any pathological lesions in terminally sacrificed rats, it is concluded that the test item did not produce any treatment related effect at the dose level used in the present study.

Interpretation of results:

other: EU GHS (CLP) criteria not met

Conclusions:

The LD50 is >2000 mg/kg bw. Based on the results of this study, an indication of the classification of the test material is as follows: EU GHS (CLP) criteria not met.

Executive summary:

In an oral toxicity study (OECD 423), two sets of fasted Wistar rats (3 femals/set) (10 - 11 weeks) were given a single oral dose of 2000 mg test material /kg bw and observed for 14 days. The conducted study was in compliance with GLP (GLP certificate issued by the Government of India, Department of Science and Technolog; National Good Laboratory Practice (GLP) Compliance Monitoring Authority (NGCMA). There were no treatment-related mortality, clinical signs and changes in body weight or necropsy findings observed so the LD50 is >2000 mg/kg bw. Based on the results of this study, an indication of the classification of the test material is as follows: EU GHS (CLP) criteria not met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The quality of the whole database is high, since a key study with zinc glucoheptonate and numerous supporting studies for zinc gluconate and other zinc compounds like zinc sulfate as well as elemental zinc are available.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The present study examined the time course and dose-response of the pulmonary injury produced by inhaled ZnO in guinea pigs, rats, rabbits, and human volunteers. The test animals were exposed to 0. 2.5, or 5.0 mg/m3 ZnO for up to 3 h and their lungs lavaged. Both the lavage fluid and recovered cells were examined for evidence of inflammation or altered cell function.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

The present study examined the time course and dose-response of the pulmonary injury produced by inhaled ZnO in guinea pigs, rats, rabbits, and human volunteers. The test animals were exposed to 0, 2.5, or 5.0 mg/m3 ZnO for up to 3 h and their lungs lavaged. Both the lavage fluid and recovered cells were examined for evidence of inflammation or altered cell function.

Species:

other: guinea pigs, rats, rabbits

Strain:

other: Hartley guinea pigs, Fischer 344 rats, New Zealand rabbits

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Male, viral antibody-free Hartley guinea pigs (300 to 400 g, Charles River Breeding Laboratories, Wilmington, Mass.), viral antibody-free Fischer 344 rats (200 to 250 g, Charles River Breeding Laboratories), and specific pathogen-free New Zealand rabbits (3 to 4 kg, Hare-Marland, Hewitt, N.J.) were used in these studies. Animals were housed in hanging, steel mesh cages and provided with water and standard laboratory animal chow (Purina, Indianapolis. Ind.) ad libitum.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

0.17 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only exposure chamber
- System of generating particulates/aerosols: Briefly, zinc granules were heated to approximately 550°C in a crucible. Zinc vapors were carried downstream by inert argon gas in the furnace to react with oxygen, yielding a supersaturated atmosphere of ZnO vapor. This vapor condensed to yield ulirafine panicles of ZnO, which were mixed with filtered air in a series of cooling/dilution heads before entering the exposure chamber.
- Method of particle size determination: Particle size measurements were performed with a differential mobility analyzer (TSI Inc., St. Paul, Minn.). By using the Hatch-Choate conversion equations,the mass median diameter of these particles was determined.

TEST ATMOSPHERE
- Brief description of analytical method used: Airborne ZnO concentrations were monitored gravimetrically with Teflon® filters (Type FG, 0.2-µm, Millipore, Bedford, Mass.) and a Cahn balance (Model 21, Cahn Instruments, Cerritos, Calif.). Periodically, filters were analyzed by atomic absorption spectroscopy for zinc content.

TEST ATMOSPHERE (if not tabulated)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The count median diameter of the particles was 0.06 µn with a geometric standard deviation of 1.8. By using the Hatch-Choate conversion equations, the mass median diameter of these particles was 0.17 µm.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: To check the 8-hr threshold limit value (TLV) of 5 mg/m3, which has been established for human exposure to ZnO fumes.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

2 - < 3 h

Remarks on duration:

2 hours (rabbit), 3 hours (guinea pigs and rats)

Concentrations:

0, 2.5 and 5 mg/m3 (lavage of lung fluid), 4.3 to 11.3 mg/m3 (determination of lung burden level) - higher concentrations of airborne ZnO were used in the lung burden studies to ensure detection of retained inhaled Zn above normal biological levels of Zn.

Control animals:

yes

Remarks:

Control animals were exposed to furnace gases consisting of air (29 L/min) and argon (1 L/min). The temperature delivered to the nose-only ports ranged from 25-30°C; relative humidity was approximately 30%.

Details on study design:

- Duration of observation period following administration: 0 - 24 h
- animals exposed to 2.5 and 5 mg/m3 ZnO: The tracheas were can-nulated sterilely with blunt needles (guinea pigs and rats) or infant endotracheal tubes (rabbits) and lavaged twice with sterile, pyrogen-free, phosphate-buffered saline (guinea pigs: 7 mL; rats: 3.5 mL; rabbits: 70% of total lung capacity). The lavage samples were saved and aliquoted for total cell counts, protein content (BioRad, Berkeley, Calif.), lactate dehydrogenase (LDH), and glucuronidase (Sigma Chemical Co., Si. Louis, Mo.). To examine functional effects of macrophages, cells in the lavage fluid from guinea pigs and rabbits were recovered by centrifugation (400 x g) for studying phagocytic function.The ability of macrophages to phagocytize particles was enumerated in two ways: (1) the phagocytic index was calculated as the percentage of macrophages engulfing particles and (2) phagocytic capacity was the percentage of viable macrophages engulfing four or more particles.
- animals exposed to 4.3 to 11.3 mg/m3 ZnO: The lungs were removed, weighed, ashed with acid, and analyzed for elemental Zn by atomic absorption spectroscopy.

Statistics:

In the animal studies, differences in lavage fluid parameters were analyzed by a one-way analysis of variance followed by a two-sided Dunnett's t-test where indicated. In the human studies, the absolute changes in lung function from pre- to postexposure values were compared by a paired t-test. A p value < 0.05 was considered statistically significant.

Sex:

male

Dose descriptor:

other: Threshold limit value

Effect level:

5 mg/m³ air

Based on:

test mat.

Exp. duration:

2 h

Remarks on result:

other: see 'Remarks'

Remarks:

a single 2- to 3-h exposure to ZnO at the current threshold limit value (TLV) of 5 mg/m3 does elicit adverse health effects in several mammalian species.

Sex:

male

Dose descriptor:

other:

Remarks:

concentration inducing pulmonary effects in guinea pigs

Effect level:

2.5 mg/m³ air

Based on:

test mat.

Exp. duration:

2 h

Remarks on result:

other: see 'Remarks'

Remarks:

Pulmonary effects in rats and guinea pigs were also observed at a concentration of 2.5 mg/m3

Mortality:

no

Other findings:

Total cell counts, protein content, LDH, and p-glucuronidase were all significantly increased in guinea pigs 24 hr after a single 3-h exposure to 5 mg/m3. Significant increases in LDH (16-fold), P-glucuronidase (5-fold), and protein were also observed in the lavage fluid of guinea pigs exposed to 2.5 mg/m3 ZnO and studied 24 h later. Rats appeared nearly as sensitive in the magnitude of their cellular and biochemical response to ZnO inhalation. Protein content, LDH, p-glucuronidase, and cell counts were increased at 24 h after exposure to 5 mg/m3. Significant increases in LDH and p-glucuronidase were also observed as early as 4 h after exposure to 5 mg/m3 and at 24 h after exposure to 2.5 mg/m3 ZnO. Inflammatory changes in the lavage fluid were not observed in rats sacrificed immediately following exposure to ZnO. No lavage parameter examined was increased in rabbits after a 2-h exposure to 5 mg/m3 ZnO. P-glucuronidase was decreased in rabbits examined at 0 and 24 hr after exposure, although this decrease cannot be explained and may be biologically irrelevant. Phagocytosis of opsonized latex beads by alveolar macrophages from guinea pigs, but not rabbits, was altered by exposure to ZnO. The phagocytic capacity of guinea pig macrophages was markedly reduced; the phagocytic index was not significantly changed.

Although the lungs of guinea pigs and rats retained approximately 20% and 12% of the inhaled dose, respectively, rabbits retained only 5%.

Conclusions:

Together, these studies demonstrate that a single 2- to 3-h exposure to ZnO at the current threshold limit value (TLV) of 5 mg/m3 does elicit adverse health effects in several mammalian species.

Executive summary:

The present study examined the time course and dose-response of the pulmonary injury produced by inhaled ZnO in guinea pigs, rats and rabbits. The test animals were exposed to 0, 2.5, or 5.0 mg/m3 ZnO for up to 3 h and their lungs lavaged. Both the lavage fluid and recovered cells were examined for evidence of inflammation or altered cell function. The lavage fluid from guinea pigs and rats exposed to 5 mg/m3 had significant increases in total cells, lactate dehydrogenase, b-glucuronidase, and protein content. These changes were greatest 24 h after exposure. Guinea pig alveolar macrophage function was depressed as evidenced by in vitro phagocytosis of opsonized latex beads. Significant changes in lavage fluid parameters were also observed in guinea pigs and rats exposed to 25 mg/m3 ZnO. In contrast, rabbits showed no increase in biochemical or cellular parameters fallowing a 2-h exposure to 5 mg/m3 ZnO. Differences in total lung burden of ZnO, as determined in additional animals by atomic absorption spectroscopy, appeared to account for the observed differences in species responses. Although the lungs of guinea pigs and rats retained approximately 20% and 12% of the inhaled dose, respectively, rabbits retained only 5%.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the present study, the effects of nasal sprays of Fe2O3 (-30 nm) and ZnO (-20 nm) nanoparticles were examined in rats to identify the nature of acute inhalation toxicity.

GLP compliance:

not specified

Specific details on test material used for the study:

Fe2O3 (ca. 30 nm) and ZnO (ca. 20 nm) nanoparticles were obtained from the Institute of High Energy Physics Chinese Academy of Sciences. The sizes of the nanoparticles were confirmed by atomic force microscopy (AFM, Shimadzu, Japan). The purities of both nanoparticie preparations were analyzed by inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7, Thermo Electron Co.). X-ray diffraction (XRD, MSAL-XD2, China) was used to characterize the structural fingerprints of the nanoparticles.

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vitalriver Experimental Animal Technology Co. Ltd (Beijing, China).
- Females (if applicable) nulliparous and non-pregnant: [yes/no] - no as only male Wistar rats are used in the study
- Age at study initiation: adult
- Weight at study initiation: 140-160 g
- Housing: kept in plastic cages
- Diet (e.g. ad libitum): a commercial pellet diet and allowed to access ad libitum
- Water (e.g. ad libitum): deionized water ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±2 °C
- Humidity (%): 50-70%
- Photoperiod (hrs dark / hrs light): 12 h light/dark rhythm

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

other: nasal sprays

Vehicle:

not specified

Mass median aerodynamic diameter (MMAD):

ca. 20 nm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE
- Exposure apparatus: sprayed directly into both nasal passages using a dry powder sprayer

ZnO and Fe2O3 nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer. Nanoparticles were placed into a sprayer and then transferred to the rat with a length of flextubing inserted into the rat nostrils. The daily administration doses were 8.5 mg/kg body weight for Fe2O3 and 2.5 mg/kg body weight for ZnO nanoparticles.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Average size of ZnO was confirmed to be 20 ± 10 nm. The shape of ZnO nanoparticles was cambiform. The purity of both nanoparticie preparations was more than 99%. Sodium & chlorine content was < 0.001%

Remarks on duration:

ZnO nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer

Concentrations:

2.5 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

yes

Remarks:

The rats in the control group were nasally sprayed with ambient air.

Details on study design:

ZnO and Fe2O3 nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer. Nanoparticles were placed into a sprayer and then transferred to the rat with a length of flextubing inserted into the rat nostrils. The daily administration doses were 2.5 mg/kg body weight for ZnO nanoparticles. Administering the nanoparticles via a dry powder sprayer coordinated with the respiration of animals has a higher delivery efficiency.
At 12 h and 36 h after the final administration, five rats from each group were sacrificed. Blood samples were taken from an abdominal vein. The brain, olfactory bulb (OB), trachea, lungs, liver, and kidney were collected. Small pieces of each tissue were fixed in 10% formalin for histopathological examination. The remaining tissues were prepared for the determination of Zn content.

Statistics:

Results were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS 11.0 (SPSS Inc., Chicago, USA). A one-way analysis of variance (ANOVA) following the least significant difference (LSD) test was used to compare all groups of animals in the study. A value of P < 0,05 was considered statistically significant.

Sex:

male

Dose descriptor:

other: see 'Remarks'

Remarks:

concentration inducing symptons

Effect level:

2.5 other: mg/kg bw

Based on:

test mat.

Remarks on result:

other: see 'Remarks'

Remarks:

After administration of 2.5 mg/kg bw ZnO nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed.

Mortality:

No deaths occurred over the entire duration of the experiment.

Clinical signs:

other: After administration of Fe2O3 and ZnO nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed in both Fe2O3-treated and ZnO-treated groups.

Animal Health

After administration of Fe2O3 and ZnO nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed in both Fe2O3-treated and ZnO-treated groups. However, no deaths occurred over the entire duration of the experiment.

The Content of Fe and Zn in Tissues

The content of Zn and Fe in lung, liver, kidney, brain, and olfactory buib tissues were quantitatively measured using NAA. Fe content in lung and liver tissues were significantly increased in the -treated group after 36 h.

Biochemical Parameters in Serum

The activities of serum ALT, AST, CK, and LDH in both the Fe2O3 -treated group were significantly lower compared to those of the control group (p<0.05) indicated the possibility of liver inflammation from this treatment.

Table 1:  Effects of Fe2O3 and ZnO nanoparticles on serum encyme levels (Mean ± S.D.).
Groups	ALT (U/L)	AST (U/L)	AST/ALT	ALP (U/L)	TP(g/L)	ALB (g/L)	GLβ (g/L)	A/G	CK (U/L)	LDH (U/L)
Control (n = 10)	47 ± 8	123 ± 28	2.6 ± 0.3	649 ± 118	49 ± 1	30 ± 1	19 ± 1	1.6 ± 0 1	1231 ± 348	990 ±383
Fe2O3 - 12 h (n=5)	44 ± 5^a	96 ± 21	2.2 ± 0.3^d	585 ± 60	52 ± 1^ad	32 ± 1^a	20 ± 0.3^ad	1.6 ± 0 1^a	671 ± 92^d	609 ± 299
Fe2O3 - 36 h (n=5)	38 ± 3^d	77 ± 5^d	2.1 ± 0.1^bd	522 ± 63	53 ± 2^bd	32 ± 2^b	21 ± 1^d	1.5 ± 0 1^cd	534 ± 111^d	224 ± 47^d
ZnO - 12 h (n=5)	33 ± 4^de	74 ± 7^d	2.3 ± 0.1	462 ± 92^d	46 ± 2^ad	27 ± 2^ad	19 ± 1^e	1.4 ± 0.1^d	616 ± 146^d	555 ± 221
ZnO - 36 h (n=5)	45 ± 4	80 ± 9^d	1.8 ± 0.04^de	527 ± 49	47 ± 2	27 ± 1^d	20 ± 2	1.4 ± 0.1^d	582 ± 181^d	353 ± 182^d
^ap< 0.05, ZnO post-exposure 12 h versus Fe2O3post-exposure 12h;
^bp< 0.05 ZnO post-exposure 36 h versus Fe2O3post-exposure 36 h,
^cp< 0.05 ZnO post-exposure 12 h versus Fe2O3post-exposure 36 h,
^dp< 0.05 ZnO/Fe2O3-treated groups versus control group.
^ep < 0.05 ZnO/Fe2O3post-exposure 12 h versus 36 h.

Histopathological Evaluation

Exposure to ZnO nanoparticles caused serious hepatic lesions compared to the controls. Inflammation, interstitial hyperemia, fatty degeneration around central vein, and hepatocyte necrosis were noticeable. In general, the pathological changes in liver were more severe in the ZnO-treated group compared to those in Fe2O3 treated group. The pathological lesions of lung tissues show inflammation, interstitial hyperemia, emphysema, and also interstitial substance hyperplasia was evident following exposure to either nanoparticie type. The lung lesions tended to be more severe in the Fe2O3 -treated group. There were no abnormal pathological changes in the trachea, kidney, or brain following administration of either type of nanoparticle.

Table 2 Pathological alterations in rats exposed to Fe2O3and ZnO nanoparticles
Groups	Liver	Kidney	Lung	Trachea
Fe2O3(12 h)
1	+^a	-	++^c	-
2	+^b	-	++^cd	-
3	+^a	-	++^c	-
Fe2O3(36 h)
1	++^f	-	++^cd	-
2	++^b	-	++^c	-
3	+^a	-	++^c	-
ZnO (12 h)
1	++^f	-	+^c	-
2	+^a	-	++^c	-
3	++^e	-	+^c	-
ZnO (36 h)
1	+^a	-	+^c	-
2	++^a	-	+^cd	-
3	++^a	-	+^c	-
Control	-	-	-	-
+ Abnormal
- Normal
^aInflammation and obvious interstitial hyperemia in liver
^bFatty degeneration of hepatocytes around central vein.
^cInflammation, obvious interstitial hyperemia and emphysema in lung.
^dInterstitial substance accrementition in lung
^eSpotty necrosis of hepatocytes
^fLarge-area necrosis of hepatocytes

Executive summary:

The toxic effects of inhalation exposure to ferric oxide (Fe₂O₃) and zinc oxide (ZnO) nanoparticles in rats were investigated. Male Wistar rats were consecutively treated with Fe₂O₃ at 8.5 mg/kg body weight and ZnO nanoparticles at 2.5 mg/kg body weight, twice daily tor 3 days. ZnO and Fe2O3 nanoparticles were sprayed directly into both nasal passages twice daily using a dry powder sprayer. Content of Fe₂O₃ and ZnO in tissues, biochemical parameters in serum, and histopathological examinations were analyzed at 12 h and 36 h after the 3 day treatment. An extended set of biochemical parameters was measured in serum, which was obtained from an abdominal vein at sacrifice. Blood serum was collected for determination of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), albumin (ALB), and globulin (GLB) by an automalic biochemical analyzer (7170A, Hitachi, Tokyo). The content of Fe and Zn in lung, liver, kidney, olfactory bulb, and brain tissues were determined by neutron activation analysis (NAA). Changes in tissue pathology were also investigated to obtain information on the range of pathological changes that might occur following acute exposure to Fe₂O₃ and ZnO nanoparticles.

After administration of Fe₂O₃ and ZnO nanoparticles for 3 days, symptoms of debilitation, anorexia, and coat dullness were observed in both Fe₂O₃-treated and ZnO-treated groups. However, no deaths occurred over the entire duration of the experiment. In the ZnO-treated group, zinc content in liver tissues was significantly increased at 12 h and further increased at 36 h. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), creatine kinase (CK), and lactate dehydrogenase (LDH) in both nanoparticle-exposed groups were significantly decreased compared to the unexposed controls. Histopathological examination showed lhat both types of nanoparticles caused severe damage in liver and lung tissues. Inflammation, interstitial hyperemia, fatty degeneration around central vein, and hepatocyte necrosis were noticeable in the liver. Inflammation, interstitial hyperemia, emphysema, and interstitial substance hyperplasia were evident in the lung. There were no abnormal pathological changes in the trachea, kidney, or brain. Although this damage progressed in both liver and lung throughout the postexposure period, no significant elevation of serum enzyme activities was observed in response to either nanoparticie type.

Once ZnO nanoparticles entered the body via inhalation, it became systemically available and caused toxic effects in internal organs other than in the lungs. Pulmonary retention, extrapulmonary translocation, and redistribution were considered to be the essential mechanisms of organ damage induced by inhaled nanoparticles. Obvious lesions of liver and lung were induced and the levels of serum ALT, AST, ALP, CK, and LDH were all significantly decreased compared with the control groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

oral route

Supporting data

As supporting information, read-across on gluconates and derivatives and zinc compounds was performed within the frame of a weight-of-evidence approach.The underlying hypothesis for the read-across is that glucoheptonates and gluconates, structurally similar sugar-like carbohydrate metal-complexes, share the same metabolism pathways in mammals (they are oxidized by pentose phosphate pathway) and that their possible toxicity is a function of the metal cation rather than of the gluconate or glucoheptonate anion.

Zinc gluconate

In general, the data on zinc gluconate allows estimating a corresponding LD50 for zinc glucoheptonate providing that no toxicity is attributed to gluconate or glucoheptonate ion, that the absorption of zinc from this zinc compounds is 100 % and all zinc became systemically available. 

 

An oral toxicology study is conducted with rats by Ash & Ash (2004). An LD50 > 5000 mg/kg bw for rats is reported, which corresponds to a LD50 > 9058.6 mg/kg bw for zinc glucoheptonate.

 

RTECS Number ZH3750000 reports an LD50 of 1290 mg/kg bw for zinc gluconate for rats, which corresponds to a LD50 of 2337.1 mg/kg bw for zinc glucoheptonate.

 

EFSA (2006) and EC (2003) reviewed publications concerning the toxic effects of dietary zinc in rats. In rats, dietary zinc intakes up to 1 g/kg body weight have been well tolerated (Kulwichet al, 1953; Sutton and Nelson, 1937; Whanger and Weswig, 1971), but dietary zinc intakes above 2 g/kg body weight have usually led to death (Sadasivan, 1951; Smith and Larson, 1946).Therefore, 2 g/kg bw correspond to the LD100 value and a concentration of 1g/kg bw is regarded as a dose that is tolerated well.

The reported LD100 and the well tolerated concentration for elemental zinc correspond to a LD100 of 12.6 g/kg bw and a well tolerated concentration of 6.3 g/kg bw for zinc glucoheptonate.

 

Salgueiro et al (2000) doses five groups of rats which consisted of 10 female and 10 male rats, each, with BioZn-AAS (zinc gluconate stabilized with glycine) at different concentrations. The tested concentrations were in the range from 1600 to 2400 mg/kg bw. The LD50 for the female rats was 2000 mg/kg bw, with a lower limit of 1810 mg/kg bw and an upper limit of 2210 mg/kg bw. For male rats, the LD50 was 1900 mg/kg bw, with a lower limit of 1756 mg/kg bw and an upper limit of 2055 mg/kg bw.

The LD50 (rat) for zinc gluconate correspond to LD50 of 3442.3 (male rats) and 3623.4 mg/kg bw (female rats) for zinc glucoheptonate.

Lewis & Kokan (1988) reported that a 17-year-old male ingested 80 to 85 zinc gluconate tablets (Lozenges) with the intention to harm himself. The zinc gluconate content per tablet was 50 mg which corresponds to a total intake of approximately 570 mg elemental zinc. He experienced severe nausea and vomiting within 30 minutes of the ingestion but without the severe caustic effects and systemic symptoms reported with ingestion of other zinc compounds related to elevated serum zinc. Serum zinc level was 4.97 mg/dL at approximately 5 hours postingestion. The relatively low percentage of zinc in zinc gluconate tablets may be a protective factor, and the binding agents in the pharmaceutical preparation may reduce the release of free zinc. Due to the body weight of 83.4 kg, the TDLo (Lowest published toxic dose) is calculated to be 47 mg/kg bw.

The TDLo for zinc gluconate is reported to be 47 mg/kg bw which corresponds to a TDLo of 296.7 mg/kg bw for zinc glucoheptonate.

 

The influence of zinc on cold as well as on Fe and Cu status within humans is well studied: Turner & Cetnarowski (2000) conducted two clinical trials, one involving 273 subjects with experimental rhinovirus colds and the other involving 281 subjects with natural colds. Volunteers received oral lozenges containing zinc gluconate (13.3 mg), zinc acetate (5 or 11.5 mg), or placebo. Zinc gluconate had no effect on symptom severity and zinc acetate had no effect on either duration or severity. Neither formulation had an effect on the duration or severity of natural cold symptoms.

Eby et al (1984) tested zinc gluconate lozenges in a double-blind, placebo controlled, clinical trial. One 23 mg zinc lozenge or matched placebo was dissolved in the mouth every 2 wakeful h after an initial double dose. Emetic properties of zinc appeared in two subjects after they ingested the loading dose. Nausea was also reported after the first few tablets. Vomiting and nausea were reported to be preventable by prior ingestion of food or drink. After 7 days, 86% of 37 zinc-treated subjects were asymptomatic, compared with only 46% of 28 placebo-treated subjects. Zinc lozenges shortened the average duration of common colds by about 7 days.

 

Fischer et al (1984) studies the effects of zinc supplementation on the copper status of healthy adult men, as assessed by the activities of the copper-metalloenzymes, plasma ferroxidase (ceruloplasmin), and erythrocyte Cu,Zn-superoxide dismutase, were determined. The subjects were given either two daily doses of 25 mg zinc or placebo for 6 weeks. No significant differences in the plasma copper levels or the ferroxidase activities between the supplemented and control groups could be detected at 2, 4, or 6 week. Plasma zinc increased and erythrocyte Cu,Zn-superoxide dismutase decreased in the supplemented group, the difference between the groups becoming significant at 6 week (p < 0.05).This suggested that the zinc supplements decreased the copper status of the experimental group.

Response of iron, copper, and zinc status to supplementation with Zn or a combination of Zn and Fe was assessed in adult females in a 10-week study by Yadrick et al (1989). The group which received 50 mg Zn/d showed significantly lower (p < 0.05) serum ferritin, hematocrit, and erythrocyte Cu,Zn-superoxide dismutase (ESOD) after 10 week supplementation compared with pretreatment levels. Serum Zn increased (p < 0.01) but no change occurred in serum ceruloplasmin, hemoglobin, or salivary sediment Zn with treatment. Within the group receiving 50 mg Fe as ferrous sulfate monohydrate in addition to 50 mg zinc ESOD decreased with treatment as did salivary sediment Zn (p < 0.05). The latter is a possible indicator suggesting that Fe possibly has an inhibitory effect on Zn status. Serum ferritin and serum Zn increased significantly, but hemoglobin, hematocrit, and ceruloplasmin were not affected by this treatment. Supplementation with Zn poses a risk to Fe and Cu status. Inclusion of Fe with Zn ameliorates the effect on Fe but not on Cu status.

 

The analysis of existing metabolism data of gluconate and glucoheptonate moieties allow to conclude that toxicity effects of the target substances glucoheptonate complexes Zn can be predicted by using the toxicological data of structurally related gluconate complexes with the same metals.Therefore, these results of Fischer et al (1984), Eby et al (1984) Turner & Cetnarowski (2000) and Yadrick et al (1989) are also relevant for zinc glucoheptonate.

 

Other zinc compounds

The risk assessment report (RAR) of 2004 summarizes LD50 values for different types of zinc sulfate. Zinc Sulfate is used as test material, however, the type of zinc sulfate is not specified in each of the reviewed studies.

LD50 values of 926 (zinc sulfate + 2 H2O) and 1.891 mg/ kg bw (zinc sulfate anhydrous) were reported by the reviewed studies for mice. The LD50 values of 926 mg/kg bw and 1891 mg/kg bw correspond to LD50 of 1935.6 and 4835.0 mg/kg bw for zinc glucoheptonate, respectively.

 

For rats, LD50 values between 920 and 2,949 mg/ kg bw were reported by the reviewed studies. Two LD50 (rat) for zinc sulfate + 7 H2O are reported to be >1000 and 2280 mg/kg bw for rats which correspond to LD50 of >1275.5 and 2908.2 mg/kg bw for zinc glucoheptonate. Additional a LD50 (rat) for zinc sulfate + 2 H2O is reported: 1710 mg/kg bw. This LD50 corresponds to a LD50 of 3574.3 mg/kg bw for zinc glucoheptonate. A LD50 (rat) of 920 mg/kg bw is reported for the unspecific zinc sulfate. It is assumed that the test material is anhydrous zinc sulfate (=worst case assumption) which corresponds to a LD50 of 2352.3 mg/kg bw for zinc glucoheptonate.

 

The LD50 values of several zinc compounds are mentioned for zinc in rats and reviewed by ATSDR (2005) and WHO(2001). The acute toxicity of zinc varied, depending on the zinc salt tested, and ranged from 237 to 623 mg zinc / kg / day. A LD50 value of 237 mg zinc / kg / day was reported for zinc acetate, 623 mg zinc / kg / day was reported for zinc sulfate. The LD50 values for zinc chloride and zinc nitrate are 528 and 293 mg zinc / kg / day, respectively. "Toxic effects of zinc in rodents following short-term oral exposure include weakness, anorexia, anaemia, diminished growth, loss of hair and lowered food utilization, as well as changes in the levels of liver and serum enzymes, morphological and enzymatic changes in the brain, and histological and functional changes in the kidney. The level at which zinc produces no adverse symptoms in rats has been set at about 160 mg/kg body weight. Pancreatic changes were observed in calves exposed to high levels of dietary zinc.” The reported LD50 values for mice of zinc acetate, zinc nitrate, zinc chloride and zinc sulfate correspond to LD50 for zinc glucoheptonate of 1496.2, 1849.7, 3333.2 and 3232.9 mg/kg bw/d, respectively.

Also, LD50 values of several zinc compounds are mentioned for zinc in mice. The acute toxicity of zinc varied, depending on the zinc salt tested, and ranged from 86 to 605 mg zinc / kg / day. A LD50 value of 86 mg zinc / kg / day was reported for zinc acetate, 605 mg zinc / kg / day was reported for zinc chloride. The LD50 values for zinc sulfate and zinc nitrate are 337 and 204 mg zinc / kg / day, respectively. "Toxic effects of zinc in rodents following short-term oral exposure include weakness, anorexia, anaemia, diminished growth, loss of hair and lowered food utilization, as well as changes in the levels of liver and serum enzymes, morphological and enzymatic changes in the brain, and histological and functional changes in the kidney."

The reported LD50 values for mice of zinc acetate, zinc nitrate, zinc chloride and zinc sulfate correspond to LD50 for zinc glucoheptonate of 542.9, 1287.8, 2127.4 and 3819.3 mg/kg bw/d, respectively.

 

A double-blind randomised trial of oral zinc supplementation was carried out by Simmer et al (1991) during the last two trimesters of pregnancy. Fifty-six women at risk of delivering a small-for-gestational-age baby received either zinc supplement (22.5 mg daily) or placebo. Twenty-nine of the women were compliant. Zinc significantly reduced the incidence of intrauterine growth retardation, and most measured indices of labour and fetal health were better in the supplemented group. Side-effects were generally no more frequent in the supplemented group than in the placebo group, but the three patients with vomiting were taking Zn. No adverse effects of Zn therapy were found.

The analysis of existing metabolism data of gluconate and glucoheptonate moieties allow to conclude that toxicity effects of the target substances glucoheptonate complexes Zn can be predicted by using the toxicological data of structurally related gluconate complexes with the same metals.Therefore, these results are also relevant for zinc glucoheptonate.

Justification for classification or non-classification

On the basis of the available data, the registered substance does not require classification for lethal effects following a single exposure according to European Regulation (EC) No 1272/2008.